BAFF, inhibitors thereof and their use in the modulation of B-cell response and treatment of autoimmune disorders

ABSTRACT

The invention provides methods for treating or preventing disorders associated with expression of BAFF comprising BAFF and fragments thereof, antibodies, agonists and antagonists.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/911,777, filed Jul. 24, 2001, which claims priority to InternationalApplication No. PCT/US00/01788 filed Jan. 25, 2000, which claimspriority to U.S. Ser. No. 60/117,169 filed on Jan. 25, 1999 and U.S.Ser. No. 60/143,228 filed Jul. 9, 2001. The entire disclosures of theaforesaid patent applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of a ligand, BAFF, a β-cellactivating factor belonging to the Tumor Necrosis Family and itsblocking agents to either stimulate or inhibit the expression of B-cellsand immunoglobulins. This protein and its receptor may have anti-cancerand/or immunoregulatory applications as well as uses for the treatmentof immunosuppressive disorders such as HIV. Specifically, the ligand andits blocking agents may play a role in the development of hypertensionand its related disorders. Furthermore, cells transfected with the genefor this ligand may be used in gene therapy to treat tumors, autoimmunediseases or inherited genetic disorders involving B-cells. Blockingagents, such as recombinant variants or antibodies specific to theligand or its receptor, may have immunoregulatory applications as well.Use of BAFF as a B-cell stimulator for immune suppressed diseasesincluding for example uses for patients undergoing organ transplantation(i.e. bone marrow transplant) as well as recovering from cancertreatments to stimulate production of B-cells are contemplated. Use ofBAFF as an adjuvant and or costimulator to boast and or restore B cellslevels to approximate normal levels are also contemplated.

BACKGROUND OF THE INVENTION

The tumor-necrosis factor (TNF)-related cytokines are mediators of hostdefense and immune regulation. Members of this family exist inmembrane-anchored forms, acting locally through cell-to-cell contact, oras secreted proteins capable of diffusing to more distant targets. Aparallel family of receptors signals the presence of these moleculesleading to the initiation of cell death or cellular proliferation anddifferentiation in the target tissue. Presently, the TNF family ofligands and receptors has at least 11 recognized receptor-ligand pairs,including: TNF:TNF-R; LT-α:TNF-R; LT-α/β:LT-β-R; FasL:Fas; CD40L:CD40;CD30L:CD30; CD27L:CD27; OX40L:OX40 and 4-1BBL:4-1BB. The DNA sequencesencoding these ligands have only about 25% to about 30% identity in eventhe most related cases, although the amino acid relatedness is about50%.

The defining feature of this family of cytokine receptors is found inthe cysteine rich extracellular domain initially revealed by themolecular cloning of two distinct TNF receptors. This family of genesencodes glycoproteins characteristic of Type I transmembrane proteinswith an extracellular ligand binding domain, a single membrane spanningregion and a cytoplasmic region involved in activating cellularfunctions. The cysteine-rich ligand binding region exhibits a tightlyknit disulfide linked core domain, which, depending upon the particularfamily member, is repeated multiple times. Most receptors have fourdomains, although there may be as few as three, or as many as six.

Proteins in the TNF family of ligands are characterized by a shortN-terminal stretch of normally short hydrophilic amino acids, oftencontaining several lysine or arginine residues thought to serve as stoptransfer sequences. Next follows a transmembrane region and anextracellular region of variable length, that separates the C-terminalreceptor binding domain from the membrane. This region is sometimesreferred to as the “stalk”. The C-terminal binding region comprises thebulk of the protein, and often, but not always, contains glycosylationsites. These genes lack the classic signal sequences characteristic oftype I membrane proteins, type I membrane proteins with the C terminuslying outside the cell, and a short N-terminal domain residing in thecytoplasm. In some cases, e.g., TNF and LT-α, cleavage in the stalkregion can occur early during protein processing and the ligand is thenfound primarily in secreted form. Most ligands, however, exist in amembrane form, mediating localized signaling.

The structure of these ligands has been well-defined by crystallographicanalyses of TNF, LT-α, and CD40L. TNF and lymphotoxin-I (LT-1) are bothstructured into a sandwich of two anti-parallel β-pleated sheets withthe “jelly roll” or Greek key topology. The rms deviation between the Cαand β residues is 0.61 C, suggesting a high degree of similarity intheir molecular topography. A structural feature emerging from molecularstudies of CD40L, TNF and LT-α is the propensity to assemble intooligomeric complexes. Intrinsic to the oligomeric structure is theformation of the receptor binding site at the junction between theneighboring subunits creating a multivalent ligand. The quaternarystructures of TNF, CD40L and LT-α have been shown to exist as trimers byanalysis of their crystal structures. Many of the amino acids conservedbetween the different ligands are in stretches of the scaffold β-sheet.It is likely that the basic sandwich structure is preserved in all ofthese molecules, since portions of these scaffold sequences areconserved across the various family members. The quaternary structuremay also be maintained since the subunit conformation is likely toremain similar.

TNF family members can best be described as master switches in theimmune system controlling both cell survival and differentiation. OnlyTNF and LTα are currently recognized as secreted cytokines contrastingwith the other predominantly membrane anchored members of the TNFfamily. While a membrane form of TNF has been well-characterized and islikely to have unique biological roles, secreted TNF functions as ageneral alarm signaling to cells more distant from the site of thetriggering event. Thus TNF secretion can amplify an event leading to thewell-described changes in the vasculature lining and the inflammatorystate of cells. In contrast, the membrane bound members of the familysend signals though the TNF type receptors only to cells in directcontact. For example T cells provide CD40 mediated “help” only to thoseB cells brought into direct contact via cognate TCR interactions.Similar cell-cell contact limitations on the ability to induce celldeath apply to the well-studied Fas system.

It appears that one can segregate the TNF ligands into three groupsbased on their ability to induce cell death. First, TNF, Fas ligand andTRAIL can efficiently induce cell death in many lines and theirreceptors mostly likely have good canonical death domains. Presumablythe ligand to DR-3 (TRAMP/WSL-1) would also all into this category. Nextthere are those ligands which trigger a weaker death signal limited tofew cell types and TWEAK, CD30 ligand and LTa1b2 are examples of thisclass. How this group can trigger cell death in the absence of acanonical death domain is an interesting question and suggests that aseparate weaker death signaling mechanism exists. Lastly, there arethose members that cannot efficiently deliver a death signal. Probablyall groups can have antiproliferative effects on some cell typesconsequent to inducing cell differentiation e.g. CD40. Funakoshi et al.(1994).

The TNF family has grown dramatically in recent years to encompass atleast 11 different signaling pathways involving regulation of the immunesystem. The widespread expression patterns of TWEAK and TRAIL indicatethat there is still more functional variety to be uncovered in thisfamily. This aspect has been especially highlighted recently in thediscovery of two receptors that affect the ability of rous sacroma andherpes simplex virus to replicate as well as the historical observationsthat TNF has anti-viral activity and pox viruses encode for decoy TNFreceptors. Brojatsch et al. (1996); Montgomery et al. (1996); Smith etal. (1994), 76 Cell 959-962; Vassalli et al. (1992), 10 Immunol.411-452.

TNF is a mediator of septic shock and cachexia, and is involved in theregulation of hematopoietic cell development. It appears to play a majorrole as a mediator of inflammation and defense against bacterial, viraland parasitic infections as well as having antitumor activity. TNF isalso involved in different autoimmune diseases. TNF may be produced byseveral types of cells, including macrophages, fibroblasts, T cells andnatural killer cells. TNF binds to two different receptors, each actingthrough specific intracellular signaling molecules, thus resulting indifferent effects of TNF. TNF can exist either as a membrane bound formor as a soluble secreted cytokine.

LT-I shares many activities with TNF, i.e. binding to the TNF receptors,but unlike TNF, appears to be secreted primarily by activated T cellsand some β-lymphoblastoid tumors. The heteromeric complex of LT-α andLT-β is a membrane bound complex which binds to the LT-β receptor. TheLT system (LTs and LT-R) appears to be involved in the development ofperipheral lymphoid organs since genetic disruption of LT-β leads todisorganization of T and B cells in the spleen and an absence of lymphnodes. The LT-β system is also involved in cell death of someadenocarcinoma cell lines.

Fas-L, another member of the TNF family, is expressed predominantly onactivated T cells. It induces the death of cells bearing its receptor,including tumor cells and HIV-infected cells, by a mechanism known asprogrammed cell death or apoptosis. Furthermore, deficiencies in eitherFas or Fas-L may lead to lymphoproliferative disorders, confirming therole of the Fas system in the regulation of immune responses. The Fassystem is also involved in liver damage resulting from hepatitis chronicinfection and in autoimmunity in HIV-infected patients. The Fas systemis also involved in T-cell destruction in HIV patients. TRAIL, anothermember of this family, also seems to be involved in the death of a widevariety of transformed cell lines of diverse origin.

CD40-L, another member of the TNF family, is expressed on T cells andinduces the regulation of CD40-bearing B cells. Furthermore, alterationsin the CD40-L gene result in a disease known as X-linked hyper-IgMsyndrome. The CD40 system is also involved in different autoimmunediseases and CD40-L is known to have antiviral properties. Although theCD40 system is involved in the rescue of apoptotic B cells, innon-immune cells it induces apoptosis. Many additional lymphocytemembers of the TNF family are also involved in costimulation.

Generally, the members of the TNF family have fundamental regulatoryroles in controlling the immune system and activating acute host defensesystems. Given the current progress in manipulating members of the TNFfamily for therapeutic benefit, it is likely that members of this familymay provide unique means to control disease. Some of the ligands of thisfamily can directly induce the apoptotic death of many transformed cellse.g. LT, TNF, Fas ligand and TRAIL. Nagata (1997) 88 Cell 355-365. Fasand possibly TNF and CD30 receptor activation can induce cell death innontransformed lymphocytes which may play an immunoregulatory function.Amakawa et al. (1996) 84 Cell 551-562; Nagata (1997) 88 Cell 355-365;Sytwu et al. (1996); Zheng et al. (1995) 377 Nature 348-351. In general,death is triggered following the aggregation of death domains whichreside on the cytoplasmic side of the TNF receptors. The death domainorchestrates the assembly of various signal transduction componentswhich result in the activation of the caspase cascade. Nagata (1997) 88Cell 355-365. Some receptors lack canonical death domains, e.g. LTbreceptor and CD30 (Browning et al. (1996); Lee et al. (1996)) yet caninduce cell death, albeit more weakly. It is likely that these receptorsfunction primarily to induce cell differentiation and the death is anaberrant consequence in some transformed cell lines, although thispicture is unclear as studies on the CD30 null mouse suggest a deathrole in negative selection in the thymus. Amakawa et al. (1996) 84 Cell551-562. Conversely, signaling through other pathways such as CD40 isrequired to maintain cell survival. Thus, there is a need to identifyand characterize additional molecules which are members of the TNFfamily thereby providing additional means of controlling diseased andmanipulating the immune system.

Sjögren's syndrome (SS) is a chronic inflammatory disorder characterizedby the destruction of exocrine glands such as salivary and lacrimalglands, leading to symptoms of dry mouth (xerostomia) and eyes(keratoconjuncitivitis sicca). Jonsson et al. (2000) Sjogren's syndromein Arthritis and allied conditions 1826-1849. SS is regarded as anautoimmune disease characterized by the presence of large mononuclearcell infiltrates in exocrine glands, B cell hyper-reactivity and variousserum autoantibodies. Jonsson et al. (2000); Manoussakis et al. (1998)Sjogren's sydrome in The autoimmune diseases, Academic Press, 381-404.SS can develop alone or in association with other autoimmune disorderssuch as systemic lupus erythematosus (SLE) and rheumatoid arthritis(RA). Jonnson et al. (2000); Manoussakis et al. (1998).

Abnormal B cell activity is a predominant feature of SS, which ismanifested by massive polyclonal B cell activation and elevatedsecretion of autoantibodies such as rheumatoid factors (RF), anti-Ro(SS-A), anti-La (SS-B) and anti-λ-fodrin autoantibodies. Jonnson et al.(2000); Manoussakis et al. (1998); MacSween et al. (1967) Ann. Rheum.Dis. 26:402-411; Haneji et al. (1997) Science 276; 604-607. Intense Bcell activity such as germinal center reactions occur in exocrine glandsof some patients, placing them in a high risk category for thedevelopment of lymphomas. Jonsson et al. (2000); Stott et al. (1998) J.Clin. Invest. 102:938-946. However, the role of B cells andautoantibodies in the pathogenesis of SS still remains unclear.

SUMMARY OF THE INVENTION

Here we characterize the functional properties of a new ligand of theTNF cytokine-family. The new ligand, termed BAFF (B cell activatingfactor belonging to the TNF family), appears to be expressed by T cellsand dendritic cells for the purpose of B-cell co-stimulation and maytherefore play an important role in the control of B cell function. Inaddition, we have generated transgenic mice overexpressing BAFF underthe control of a liver-specific promoter. These mice have excessivenumbers of mature B cells, spontaneous germinal center reactions,secrete autoantibodies, and have high plasma cell numbers in secondarylymphoid organs and Ig deposition in the kidney.

The BAFF Tg mice develop as they age a secondary condition to theirlupus-like disease, showing interesting similarities with that of SS inhumans. We also identified a new and potentially pathogenic B cellpopulation with MZ-like features infiltrating salivary glands of BAFF Tgmice.

Accordingly, the present invention is directed to the use ofBAFF-ligands, blocking agents and antibodies for the ligand, to eitherstimulate or inhibit the growth of B-cells and the secretion ofimmunoglobulin. The claimed invention may be used for therapeuticapplications in numerous diseases and disorders, as discussed in moredetail below, as well as to obtain information about, and manipulate,the immune system and its processes. Further, this invention can be usedas a method of stimulating or inhibiting the growth of B-cells and thesecretion of immunoglobulins. BAFF associated molecules, as described bythis invention, may also have utility in the treatment of autoimmunediseases, disorders relating to B-cell proliferation and maturation,BAFF ligand regulation and inflammation. The invention may be involvedin the regulation or prevention of hypertension and hypertension-relateddisorders of the renal and cardiovascular tissue.

Additional features and advantages of the invention will be set forth inthe description which follows, and in part will be apparent from thedescription, or may be learned by practice of the invention. Theobjectives and other advantages of the invention will be realized andattained by the methods particularly pointed out in the writtendescription and claims hereof, as well as in the appended drawings.

Thus, to achieve these and other advantages, and in accordance with thepurpose of the invention, as embodied and broadly described herein, theinvention includes a method of effecting B-cell growth and secretion ofimmunoglobulins through the administration of various BAFF ligands andrelated molecules.

The invention also contemplates stimulating B-cell growth through theuse of BAFF ligands or active fragments of the polypeptide. Thepolypeptide may be use alone or with a CD40 ligand or an anti-murineantibody.

In other embodiments, the invention relates to methods of stimulation ofdendritic cell-induced B-cell growth and maturation through the use ofBAFF ligands or active fragments of BAFF. Again, the polypeptide may beused alone or with CD40 ligand or anti-μ antibodies.

In other embodiments, blocking agents of BAFF and the BAFF receptor havebeen used to inhibit B-cell growth and immunoglobulin secretion. Theseagents can be inoperative, recombinant BAFF, BAFF specific antibodies,BAFF-receptor specific antibodies or an anti-BAFF ligand molecule.

In yet other embodiments, the invention relates to the use of BAFF, BAFFrelated molecules and BAFF blocking agents to treat hypertension,hypertension related disorders, immune disorders, autoimmune diseases,inflammation and B-cell lympho-proliferate disorders. In another aspect,the invention relates to the use of BAFF, BAFF-related molecules andBAFF blocking agents to treat Sjogren's syndrome.

In a preferred embodiment, the invention relates to methods for treatingor reducing the advancement, severity or effects of Sjogren's syndromein a patient by administering a pharmaceutical preparation comprising atherapeutically effective amount of a BAFF blocking agent and apharmaceutically acceptable carrier. In some embodiments, the BAFFblocking agent may be a soluble BAFF receptor molecule, an antibodydirected against BAFF-ligand or an antibody directed against a BAFFreceptor. The BAFF receptor may be BCMA, TACI, or BAFF R.

The invention encompasses the use of BAFF and BAFF-related molecules aseither agonists or antagonists in effecting immune responses byeffecting the growth and/or maturation of B-cells and secretion ofimmunoglobulin.

The invention relates in other embodiments to soluble constructscomprising BAFF which may be used to directly trigger BAFF mediatedpharmacological events. Such events may have useful therapeutic benefitsin the treatment of cancer, tumors or the manipulation of the immunesystem to treat immunologic diseases.

Additionally, in other embodiments the claimed invention relates toantibodies directed against BAFF ligand, which can be used, for example,for the treatment of cancers, and manipulation of the immune system totreat immunologic disease.

In yet other embodiments the invention relates to methods of genetherapy using the genes for BAFF.

The pharmaceutical preparations of the invention may, optionally,include pharmaceutically acceptable carriers, adjuvants, fillers, orother pharmaceutical compositions, and may be administered in any of thenumerous forms or routes known in the art.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory, andare intended to provide further explanation of the invention as claimed.

The accompanying drawings are included to provide a furtherunderstanding of the invention, and are incorporated in, and constitutea part of this specification, illustrate several embodiments of theinvention, and together with the description serve to explain theprinciples of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) depicts the predicted amino acid sequence of human [SEQ. ID.NO.: 1] and mouse BAFF [SEQ. ID. NO.:2]. The predicted transmembranedomain (TMD, dashed line), the potential N-linked glycosylation sites(stars) and the natural processing site of human BAFF (arrow) areindicated. The double line above hBAFF indicates the sequence obtainedby Edman degradation of the processed form of BAFF. (B) Depicts acomparison of the extracellular protein sequence of BAFF [SEQ. ID. NO.:3] and some members of the TNF ligand family [SEQ. ID. NO.: 4 (hAPRIL);SEQ. ID. NO.: 5 (hTNF alpha); SEQ. ID. NO.: 6 (hFasL); SEQ. ID. NO.: 7(hLT alpha); SEQ. ID. NO.: 8 (hRANKL)]. Identical and homologousresidues are represented in black and shaded boxes, respectively. (C)Depicts dendrogram of TNF family ligands.

FIG. 2 is a schematic characterization of recombinant BAFF (A) Schematicrepresentation of recombinant BAFF constructs. Soluble recombinant BAFFsstarting at Leu₈₃ and Gln₁₃₆ are expressed fused to a N-terminal Flagtag and a 6 amino acid linker. The long form is cleaved between Arg₁₃₃and Ala₁₃₄ (arrow) in 293 T cells, to yield a processed form of BAFF.Asn₁₂₄ and Asn₂₄₂ belong to N-glycosylation consensus sites. N-linkedglycan present on Asn₁₂₄ is shown as a Y. TMD: transmembrane domain. (B)Peptide N-glycanase F (PNGase F) treatment of recombinant BAFF.Concentrated supernatants containing Flag-tagged BAFFs and APRIL weredeglycosylated and analyzed by Western blotting using polyclonalanti-BAFF antibodies or anti-Flag M2, as indicated. All bands exceptprocessed BAFF also reacted with anti-Flag M2 (data not shown). (C) Fulllength BAFF is processed to a soluble form. 293T cells were transientlytransfected with full length BAFF. Transfected cells and theirconcentrated supernatants were analyzed by Western blotting usingpolyclonal anti-BAFF antibodies. Supernatants corresponding to 10× theamount of cells were loaded onto the gel. (D) Size exclusionchromatography of soluble BAFF on Superdex-200. Concentratedsupernatants containing soluble BAFF/short were fractionated on aSuperdex-200 column and the eluted fractions analyzed by Westernblotting using anti-Flag M2 antibody. The migration positions of themolecular mass markers (in kDa) are indicated on the left-hand side forSDS-PAGE and at the top of the figure for size exclusion chromatography.

FIG. 3 depicts expression of BAFF (A) Northern blots (2 μg poly A⁺ RNAper lane) of various human tissues were probed with BAFF antisense mRNA.(B) Reverse transcriptase amplification of BAFF, IL-2 receptor alphachain and actin from RNA of purified blood T cells at various timepoints of PHA activation, E-rosetting negative blood cells (B cells andmonocytes), in vitro derived immature dendritic cells, 293 cells, and293 cells sterilely transfected with full length BAFF (293-BAFF).Control amplifications were performed in the absence of added cDNA. IL-2receptor alpha chain was amplified as a marker of T cell activation.

FIG. 4 depicts BAFF binding to mature B cells. (A) Binding of solubleBAFF to BJAB and Jurkat cell lines, and to purified CD19⁺ cells of cordblood. Cells were stained with the indicated amount (in ng/50 μl) ofFlag-BAFF and analyzed by flow cytometry. (B) Binding of soluble BAFF toPBLS. PBLs were stained with anti-CD8-FITC or with anti-CD19-FITC(horizontal axis) and with Flag-BAFF plus M2-biotin and avidin-PE(vertical axis). Flag-BAFF was omitted in controls.

FIG. 5 depicts BAFF costimulates B cell proliferation. (A) Surfaceexpression of BAFF in stably transfected 293 cells. 293-BAFF and 293wild-type cells were stained with anti-BAFF mAb 43.9 and analyzed byflow cytometry. (B) Costimulation of PBLs by 293-BAFF cells. PBLs(10⁵/well) were incubated with 15.000 glutaraldehyde-fixed 293 cells(293 wt or 293-BAFF) in the presence or absence of anti-B cell receptorantibody (anti-μ). Fixed 293 cells alone incorporated 100 cpm. (C) Dosedependent costimulation of PBL proliferation by soluble BAFF in thepresence of anti-μ. Proliferation was determined after 72 h incubationby [³H]-thymidine incorporation. Controls include cells treated withBAFF alone, with heat-denatured BAFF or with an irrelevant isotypematched antibody in place of anti-μ. (D) Comparison of (co)stimulatoryeffects of sCD40L and sBAFF on PBL proliferation. Experiment wasperformed as describe in panel C. (EAFF costimulates Ig secretion ofpreactivated human B cells. Purified CD19⁺ B cells were activated bycoculture with EL-4 T cells and activated T cell supernatants for 5-6 d,then re-isolated and cultured for another 7 days in the presence ofmedium only (−) or containing 5% activated T cell supernatants (T-SUP)or a blend of cytokines (IL-2, IL-4, IL-10). The columns represent meansof Ig concentrations for cultures with or without 1 μg/ml BAFF. Means±SDin terms of “fold increase” were 1.23±0.11 for medium only, 2.06±0.18with T cell supernatants (4 experiments) and 1.45±0.06 with IL-2, IL-4and IL-10 (2 experiments). These were performed with peripheral blood (3experiments) or cord blood B cells (one experiment; 2.3 fold increasewith T cell supernatants, 1.5 fold increase with IL-2, IL-4 and IL-10).(F) Dose-response curve for the effect of BAFF in cultures with T cellsupernatants, as shown in panel D. Mean±SD of 3 experiments.

FIG. 6 depicts that BAFF acts as a cofactor for B cell proliferation.The proliferation of human PBL was measured alone (500 cpm), with thepresence of BAFF ligand alone, with the presence of goat anti-murine(mu) alone, and with both BAFF ligand and anti-mu. The combination ofboth anti-mu and BAFF significantly raised proliferation of PBL as theconcentration of BAFF increased suggesting BAFF's cofactorcharacteristics.

FIG. 7 depicts increased B cell numbers in BAFF Tg mice.

(A) Increased lymphocytes counts in BAFF Tg mice. The graph compares 12control littermates (left panel) with 12 BAFF Tg mice (right panel).Lymphocytes counts are shown with circles and granulocytes (includingneutrophils, eosinophils, basophils) with diamonds.

(B) Increased proportion of B cells in PBL from BAFF Tg mice. PBL werestained with both anti-B220-FITC and anti-CD4-PE for FACS analysis andgated on live cells using the forward side scatter. Percentages of CD4and B220 positive cells are indicated. One control mouse (left) and twoBAFF Tg mice (right) are shown and the results were representative of 7animals analysed in each group.

(C) FACS analysis of the ratio of B to T cells in PBL. The differencebetween control animals and BAFF Tg mice in (A) and (C) wasstatistically significant (P<0.001).

(D) Increased MHC class II expression on B cells from BAFF Tg mice PBL.MHC class II expression was analysed by FACS.

(E) Increased Bcl-2 expression in B cells from BAFF Tg mice PBL. Bcl-2expression was measured by intracytoplasmic staining and cells wereanalysed by FACS. In both (D) and (E) Live cells were gated on theforward side scatter. Four control littermates (white bars) and 4 BAFFTg mice are shown and are representative of at least 12 animals analysedfor each group. MFI: mean of fluorescence intensity. The differencebetween control animals and BAFF Tg mice was statistically significant(P<0.005).

(F) Increased expression of effector T cells in BAFF Tg mice. PBL werestained with anti-CD4-Cychrome, anti-CD44-FITC and anti-L selectin-PE.Are shown CD4⁺-gated cells. Percentages of CD44^(hi)/L-selectin^(lo)cells are indicated. One control mouse (left) and two BAFF Tg mice(right) are shown and the results were representative of 8 animalsanalysed in each group.

FIG. 8 depicts increased B cell compartments in the spleen but not inthe bone marrow of BAFF Tg mice.

(A) FACS staining for mature B cells using both anti-IgM-FITC andanti-B220-PE, in spleen (top panel), bone marrow (medium panel) and MLN(bottom panel). Percentages of B220+/IgM+mature B cells are indicated.

(B) FACS staining for preB cells (B220+/CD43−) and proB cells(B220+/CD43+) in the bone marrow using anti-CD43-FITC,anti-B220-Cy-chrome and anti-IgM-PE simultaneously. Are shown cellsgated on the IgM negative population. Percentages of preB cells(B220+/CD43−) and proB cells (B220+/CD43+) cells are indicated.

For all figures (A and B) one control mouse (left) and two BAFF Tg mice(right) are shown and results are representative of 7 animals analysedfor each group.

FIG. 9 depicts increased Ig, RF and CIC levels in BAFF Tg mice

(A) SDS-PAGE of two control sera (−) and 4 sera from BAFF Tg mice (+)side by side with the indicated amount of a purified mouse IgG forreference. The intensity of the albumin band in similar in all lanesindicating that the material loaded on the gel is equivalent for eachsample. ELISA-based analysis of total mouse Ig (B), RF (C) and CIC (D)in the sera of 19 control littermates (white bars) and 21 BAFF Tg mice(Black bars). In the absence of a proper RF control, the titer (log base2) for RF is defined as the dilution of the sera giving an O.D. 3 timeshigher than that of background. The quantity of CIC is defined as thequantity of PAP required to generate an O.D. equivalent to that obtainedwith the tested serum. The difference between control animals and BAFFTg mice was statistically significant (P<0.001 in (B) and (C), P<0.003in (D)).

FIG. 10 depicts the presence of anti-ssDNA and anti-dsDNA autoantibodiesin some BAFF Tg mice.

(A) Analysis by ELISA of anti-ssDNA autoantibodies in 19 controllittermates (gray bars) and 21 BAFF Tg mice (black bars).

(B) Analysis by ELISA of anti-ssDNA autoantibodies in 5 controllittermates and the 5 animals showing levels of anti-ssDNAautoantibodies from (A).

(C) Paraffin sections of kidneys from a control mouse (left) and a BAFFTg mouse (right), stained with goat anti-mouse Ig-HRP. Ig deposition isshown by a brown staining. These pictures are representative of 6 BAFFTg mice analysed.

FIG. 11 depicts enlarged Peyer's patches in BAFF Tg mice. Photography ofPeyers patches (indicated with an arrow) on the small intestine of acontrol mouse (left) and a BAFF Tg mouse (right). This pictures isrepresentative of at least 12 mice sacrificed for each group.Magnification 5×

FIG. 12 depicts disrupted T and B cell organization, intense germinalcenter reactions, decreased number of dendritic cells and increasednumber of plasma cells in the spleen of BAFF Tg mice.

A control mouse is shown in A, C, E and G and a BAFF Tg in B, D, F, andH. B cells are blue and T cells brown (A and B). Germinal centers areshown with an arrow (C and D). Only few residual germinal centers areseen in control mice (C). CD11c positive dendritic cells are brown andappear in the T cell zone, bridging channels and the marginal zone (E).Very few are present in BAFF Tg mice (F). Syndecan-1-positive plasmacells were only detectable in the red pulp of BAFF Tg mice (H) but notcontrol mice (G).

These pictures are representative of at least 12 BAFF Tg mice analysedand 12 control mice. The magnification is 100× for all pictures except Cand D which are 50×. B: B cell follicle, T: PALS, WP: white pulp, RP:red pulp.

FIG. 13 depicts disrupted T and B cells organization, intense germinalcenter reactions and large number of plasma cells in the MLN of BAFF Tgmice.

The control mouse is shown in A, C, E and G and the BAFF Tg mouse isshown in B, D, F, and H. The immunohistochemistry was performed asdescribed in FIG. 6. T and B cell staining is shown in A and B, germinalcenters in C and D, dendritic cells E and F and plasma cells in G and H.GC: germinal center. Magnification 100×.

FIG. 14 depicts enlarged and inflamed salivary glands BAFF Tg mice. (A)Mice 15-17 months old, 4 control littermates (white bar) and 4 BAFF Tgmice (grey bar) were sacrificed the same day for organ collection. Bothright and left submaxillary glands were collected and weighted. The datashows the mean t standard deviation of weight for both glands. Theseresults are representative of at least 4 separate groups of age-matchedanimals dissected. (B) Paraffin sections of submaxillary glands from acontrol littermate (left panel) and two BAFF Tg mice (middle and rightpanel) were stained with H&E. Arrows indicate ducts and acinar cells inthe left and right panels. The arrow in the middie panel is showingacinar destruction. White stars indicate periductal infiltrates (foci).Magnification 100×. (C) Paraffin sections of submaxillary glands from 7control mice and 22 BAFF Tg mice (age 12-17 months) were prepared asshown in (B) and scored for the disease as described in materials andmethods. The mean score of disease is indicated with a bar. *p<0.05,**p<0.03.

FIG. 15 depicts tumor-like clusters of B-lymphoid cells in asubmaxillary tumor or infiltrates present in submaxillary glands of BAFFTg mice. Frozen sections of submaxillary glands (A and C) or asubmaxillary tumor (B, D and E) were stained with H&E (A, B and C) orBiotin-labelled anti-mouse B220 (D) or anti-mouse syndecan-1 (E),follwed by Streptavidin-HRP brown staining in D and E). Arrows in A, B,C and D indicate the tumor-like clusters, arrow in E shows the presenceof syndecan-1 positive plasma cells whereas the stars indicate thelocation of tumor-like clusters. Note that tumor-like clusters andplasma cells do not colocalize. Panels (A) and (C) are representative of4 animals in which these clusters were detected as cells infiltratingsubmaxillary glands. Panels (B), (D) and (E) are representative of 3submaxillary tumors analysed. Magnification is indicated for each panel.A germinal center (GC) is also indicated in panel (B).

FIG. 16 depicts identification of a Mz-like B cell populationinfiltrating salivary glands of BAFF Tg mice. Submaxillary glands from13-17 months old BAFF Tg mice and age-matched control littermates weredigested with collagenase for purification of infiltrating lymphocytesas described in materials and methods. Using four-color flow cytometryanalysis, cells were stained with anti-B220, anti-CD5, anti-IgM andanti-CD43. (A) shows the gating on B220^(hi) and B220^(lo/int) B cellson control (left) and BAFF Tg mice (right) as indicated. (B) shows fourdot plots for expression of IgM and CD5 on B220^(hi) cells (left) andB220^(lo/int) (right) from BAFF Tg mice and control mice as indicated.The IgM^(hi) B cell gate on B220^(hi) cells (left) and the B-1a and B-1bgates on B220^(lo/int) cells are shown. (C) shows histograms for theexpression of CD43 on gated IgM^(hi)/B220^(hi) cells (left) and gatedB-1a and B-1b cells (right) from BAFF Tg mice as indicated. (D)Lymphocytes prepared from submaxillary glands (left panel) and inguinallymph nodes (right panel) of a BAFF Tg mouse were stained withanti-B220, anti-IgM and anti-L-selectin, using a four-color flowcytometry procedure. Cells from submaxillary glands were gated onB220^(hi)/IgM^(hi) (left panel) and on IgM+(right panel). A histogram ofL-selectin expression is shown in each panel. (E) Lymphocytes from aBAFF Tg mice (right) and a control littermate (left) were prepared as inA and stained with anti-B220, anti-IgM and either anti-IgD, anti-CD23,anti-CD21, anti-CD1, anti-HSA, using a three color flow cytometryprocedure. Cells were gated on B220^(hi) and IgM^(hi) cells as indicatedon the top histograms and expression of IgD, CD23, CD1, CD21 and HSA onthese gated cells is shown on histograms for control (left) and Tg mice(right) as indicated. In (C), (D) and (E), mean of fluorescenceintensity (MFI) and a bar delineating the area in which the negativecontrol histogram is located are indicated in each histogram. (F)Schematic representation of the common and distinct markers expressed onMZ (right circle), B-1 (left circle) and T1 B cells (lower circle). Thephenotype of the B cells infiltrating submaxillary glands of BAFF Tgmice is indicated by an oval with a dotted line. These results arerepresentative of at least 12 BAFF Tg and 7 control mice analysed.

FIG. 17 depicts decreased saliva flow in older BAFF Tg mice. 13 BAFF Tgmice (diamonds) and 14 control littermates (circles) were injected withpilocarpine prior to saliva collection as described in materials andmethods. Mice 13-15.5 months old are shown in (A) and mice 8-10 monthsold are shown in (B). Means of saliva flow are shown with a bar and pvalues are indicated in each panel.

FIG. 18 depicts elevated levels of BAFF in sera and salivary glandtissues from patients suffering from primary SS but lack of correlationof these levels with levels of total IgG, RF and presence of anti-Ro/Laautoantibodies. (A) individual BAFF levels in 39 healthy controls(square), 41 patients with primary SS (diamond), 53 patients with SLE(circle) and 53 patients with RA (triangle) were measured in sera byELISA and BAFF levels plotted on a log scale. Controls were drawn fromnormal healthy donors. The horizontal black bars indicates the averagefor each group: normal 10.4±13 (ng/ml), SS patients 53±67 (ng/ml), SLEpatients 12.7±24.4 (ng/ml) and RA patients 23±47 (ng/ml). Someindividuals did not have detectable amount of BAFF in their serum and donot appear on the log scale, this includes 16 normals, 6 SS, 20 SLE and10 RA patients. The dotted line delineates the range of normal BAFFlevels. *p<0.04, the p value was determined by ANOVA t test. (B) and (C)show the correlation of BAFF levels in the sera of patients with SS withthe corresponding levels of IgG (B) and RF (C) in each serum. r and pvalues calculated by ANOVA are shown. (D) shows the levels of BAFF inpatients with detected anti-Ro and anti-La (left), anti-Ro only (middle)or no precipitin detected (right). (E) Paraffin sections of a humanlabial salivary gland biopsy from a patient with SS were stained with arat anti-human BAFF antibody (right) or an isotype-matched controlantibody (left). Staining of normal human labial salivary gland with ratanti-human BAFF antibody is also shown (bottom left, magnification100×). The staining was revealed using biotin-labelled anti-rat Igfollowed by Streptavidin-HRP. The staining appears brown on thesections. Magnification 200×. These pictures are representative of 4patients with primary SS and 3 control tissues analysed.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the present preferredembodiments of the invention. This invention relates to the use of BAFFand BAFF related molecules to effect the growth and maturation ofB-cells and the secretion of immunoglobulin. The invention relates tothe use of BAFF and BAFF related molecules to effect responses of theimmune system, as necessitated by immune-related disorders.Additionally, this invention encompasses the treatment of cancer andimmune disorders through the use of a BAFF, BAFF blocking agents, orBAFF related gene through gene therapy methods.

The BAFF ligand and homologs thereof produced by hosts transformed withthe sequences of the invention, as well as native BAFF purified by theprocesses known in the art, or produced from known amino acid sequences,are useful in a variety of methods for anticancer, antitumor andimmunoregulatory applications. They are also useful in therapy andmethods directed to other diseases.

“BAFF blocking agents” refers to agents that can diminish BAFF ligandbinding to BAFF receptors, or can diminish BAFF receptor signalling, orthat can influence how the BAFF receptor signal is interpreted withinthe cell.

A BAFF blocking agent that acts by diminishing ligand-receptor bindingcan inhibit BAFF ligand binding by at least 20%. Examples of BAFFblocking agents include soluble BAFF receptor-Fc molecules, anti-BAFFligand antibodies and anti-BAFF receptor antibodies.

“BAFF receptors” have been identified and characterized and include TACI(see, e.g., U.S. Pat. No. 5,969,102 and WO98/39361, incorporated hereinby reference), BCMA (see, e.g., WO01/12812, incorporated herein byreference), and BAFFR (see, e.g., Thompson et al. (2001) Science293:2108, incorporated herein by reference).

Another aspect of the invention relates to the use of the polypeptideencoded by the isolated nucleic acid encoding the BAFF-ligand in“antisense” therapy. As used herein, “antisense” therapy refers toadministration or in situ generation of oligonucleotides or theirderivatives which specifically hybridize under cellular conditions withthe cellular mRNA and/or DNA encoding the ligand of interest, so as toinhibit expression of the encoded protein, i.e. by inhibitingtranscription and/or translation. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to a range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid, which, when transcribed in the cell,produces RNA which is complementary to at least a portion of thecellular mRNA which encodes Kay-ligand. Alternatively, the antisenseconstruct can be an oligonucleotide probe which is generated ex vivo.Such oligonucleotide probes are preferably modified oligonucleotideswhich are resistant to endogenous nucleases, and are therefor stable invivo. Exemplary nucleic acids molecules for use as antisenseoligonucleotides are phosphoramidates, phosphothioateandmethylphosphonate analogs of DNA (See, e.g., U.S. Pat. No. 5,176,996;U.S. Pat. No. 5,264,564; and U.S. Pat. No. 5,256,775). Additionally,general approaches to constructing oligomers useful in antisense therapyhave been reviewed, for example, by Van Der Krol et al., (1988)Biotechniues 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668, specifically incorporated herein by reference.

A. BAFF-LIGAND

The BAFF-ligand of the invention, as discussed above, is a member of theTNF family and is described in PCT application number PCT/US98/19037(WO99/12964) and is incorporated in its entirety herein. The protein,fragments or homologs thereof may have wide therapeutic and diagnosticapplications.

The BAFF-ligand is present primarily in the spleen and in peripheralblood lymphocytes, strongly indicating a regulatory role in the immunesystem. Comparison of the claimed BAFF-ligand sequences with othermembers of the human TNF family reveals considerable structuralsimilarity. All the proteins share several regions of sequenceconservation in the extracellular domain.

Although the precise three-dimensional structure of the claimed ligandis not known, it is predicted that, as a member of the TNF family, itmay share certain structural characteristics with other members of thefamily.

The novel polypeptides of the invention specifically interact with areceptor, which has not yet been identified. However, the peptides andmethods disclosed herein enable the identification of receptors whichspecifically interact with the BAFF-ligand or fragments thereof.

The claimed invention in certain embodiments includes methods of usingpeptides derived from BAFF-ligand which have the ability to bind totheir receptors. Fragments of the BAFF-ligands can be produced inseveral ways, e.g., recombinantly, by PCR, proteolytic digestion or bychemical synthesis. Internal or terminal fragments of a polypeptide canbe generated by removing one or more nucleotides from one end or bothends of a nucleic acid which encodes the polypeptide. Expression of themutagenized DNA produces polypeptide fragments.

Polypeptide fragments can also be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-moc or t-boc chemistry. For example, peptides and DNA sequences of thepresent invention may be arbitrarily divided into fragments of desiredlength with no overlap of the fragment, or divided into overlappingfragments of a desired length. Methods such as these are described inmore detail below.

B. Generation of Soluble Forms of BAFF-Ligand and BAFF-Receptors

Soluble forms of the BAFF-ligand can often signal effectively and hencecan be administered as a drug which now mimics the natural membraneform. It is possible that the BAFF-ligand claimed herein are naturallysecreted as soluble cytokines, however, if not, one can reengineer thegene to force secretion. To create a soluble secreted form ofBAFF-ligand or BAFF receptor, one would remove at the DNA level theN-terminus transmembrane regions, and some portion of the stalk region,and replace them with a type I leader or alternatively a type II leadersequence that will allow efficient proteolytic cleavage in the chosenexpression system. A skilled artisan could vary the amount of the stalkregion retained in the secretion expression construct to optimize bothreceptor binding properties and secretion efficiency. For example, theconstructs containing all possible stalk lengths, i.e. N-terminaltruncations, could be prepared such that proteins starting at aminoacids 81 to 139 would result. The optimal length stalk sequence wouldresult from this type of analysis.

Soluble forms of BAFF receptors can be prepared using techniqueswell-known to those of ordinary skill in the art. Immunoglobulin Fcportions may be fused to the BAFF receptor protein to increase thehalf-life of the soluble BAFF receptor. Production of such soluble BAFFreceptor proteins is described, for example, in WO 01/12812, WO01/24811, and PCT/US01/40626, the entire disclosures of which areincorporated herein by reference.

C. Generation of Antibodies Reactive with the BAFF-Ligand and BAFFReceptors

The invention also includes antibodies specifically reactive with theclaimed BAFF-ligand or its receptors. Anti-protein/anti-peptide antiseraor monoclonal antibodies can be made by standard protocols (See, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). A mammal such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the peptide.Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers, or other techniques, well known in the art.

An immunogenic portion of BAFF-ligand or its receptors can beadministered in the presence of an adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassays can be used with theimmunogen as antigen to assess the levels of antibodies.

In a preferred embodiment, the subject antibodies are immunospecific forantigenic determinants of BAFF-ligand or its receptors, (e.g. antigenicdeterminants of a polypeptide of SEQ. ID. NO.: 2, said sequence asdescribed in PCT application number PCTI/US98/19037 (WO99/12964) and isincorporated in its entirety herewith), or a closely related human ornon-human mammalian homolog (e.g. 70, 80 or 90 percent homologous, morepreferably at least 95 percent homologous). In yet a further preferredembodiment of the present invention, the anti-BAFF-ligand oranti-BAFF-ligand-receptor antibodies do not substantially cross react(i.e. react specifically) with a protein which is e.g., less than 80percent homologous to SEQ. ID. NO.: 2 or 6 said sequence as described inPCT application number PCT/US98/19037 (WO99/12964) and is incorporatedin its entirety herewith; preferably less than 90 percent homologouswith SEQ. ID. NO.: 2 said sequence as described in PCT applicationnumber PCT/US98/19037 (WO99/12964) and is incorporated in its entiretyherewith; and, most preferably less than 95 percent homologous with SEQ.ID. NO.: 2 said sequence as described in PCT application numberPCT/US98/19037 (WO99/12964) and is incorporated in its entiretyherewith. By “not substantially cross react”, it is meant that theantibody has a binding affinity for a non-homologous protein which isless than 10 percent, more preferably less than 5 percent, and even morepreferably less than 1 percent, of the binding affinity for a protein ofSEQ. ID. NO.: 2 said sequence as described in PCT application numberPCT/US98/19037 (WO99/12964) and is incorporated in its entiretyherewith.

Production of exemplary anti-BAFF receptor antibodies is described, forexample, in WO 01/12812, WO 01/24811 and PCT/US01/40626, the entiredisclosures of which are incorporated herein by reference.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with BAFF-ligand, or itsreceptors. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab′)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments. The antibodies of the present invention are further intendedto include biospecific and chimeric molecules having anti-BAFF-ligand oranti-BAFF-ligand-receptor activity. Thus, both monoclonal and polyclonalantibodies (Ab) directed against BAFF-ligand, Tumor-ligand and theirreceptors, and antibody fragments such as Fab′ and F(ab′)₂, can be usedto block the action of the Ligand and their respective receptor.

Various forms of antibodies can also be made using standard recombinantDNA techniques. Winter and Milstein (1991) Nature 349: 293-299,specifically incorporated by reference herein. For example, chimericantibodies can be constructed in which the antigen binding domain froman animal antibody is linked to a human constant domain (e.g. Cabilly etal., U.S. Pat. No. 4,816,567, incorporated herein by reference).Chimeric antibodies may reduce the observed immunogenic responseselicited by animal antibodies when used in human clinical treatments.

In addition, recombinant “humanized antibodies” which recognizeBAFF-ligand or its receptors can be synthesized. Humanized antibodiesare chimeras comprising mostly human IgG sequences into which theregions responsible for specific antigen-binding have been inserted.Animals are immunized with the desired antigen, the correspondingantibodies are isolated, and the portion of the variable regionsequences responsible for specific antigen binding are removed. Theanimal-derived antigen binding regions are then cloned into theappropriate position of human antibody genes in which the antigenbinding regions have been deleted. Humanized antibodies minimize the useof heterologous (i.e. inter species) sequences in human antibodies, andthus are less likely to elicit immune responses in the treated subject.

Construction of different classes of recombinant antibodies can also beaccomplished by making chimeric or humanized antibodies comprisingvariable domains and human constant domains (CH1, CH2, CH3) isolatedfrom different classes of immunoglobulins. For example, antibodies withincreased antigen binding site valencies can be recombinantly producedby cloning the antigen binding site into vectors carrying the human:chain constant regions. Arulanandam et al. (1993) J. Exp. Med., 177:1439-1450, incorporated herein by reference.

In addition, standard recombinant DNA techniques can be used to alterthe binding affinities of recombinant antibodies with their antigens byaltering amino acid residues in the vicinity of the antigen bindingsites. The antigen binding affinity of a humanized antibody can beincreased by mutagenesis based on molecular modeling. Queen et al.,(1989) Proc. Natl. Acad. Sci. 86: 10029-33 incorporated herein byreference.

D. Generation of Analogs: Production of Altered DNA and PeptideSequences

Analogs of the BAFF-ligand can differ from the naturally occurringBAFF-ligand in amino acid sequence, or in ways that do not involvesequence, or both. Non-sequence modifications include in vivo or invitro chemical derivatization of the BAFF-ligand. Non-sequencemodifications include, but are not limited to, changes in acetylation,methylation, phosphorylation, carboxylation or glycosylation.

Preferred analogs include BAFF-ligand biologically active fragmentsthereof, whose sequences differ from the sequence given in SEQ. ID NO. 2said sequence as described in PCT application number PCT/US98/19037(WO99/12964) and is incorporated in its entirety herewith, by one ormore conservative amino acid substitutions, or by one or morenon-conservative amino acid substitutions, deletions or insertions whichdo not abolish the activity of BAFF-ligand. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics, e.g. substitutions within the following groups:valine, glycine; glycine, alanine; valine, isoleucine, leucine; asparticacid, glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and, phenylalanine, tyrosine.

E. Materials and Methods of the Invention

The anti-Flag M2 monoclonal antibody, biotinylated anti-Flag M2 antibodyand the anti-Flag M2 antibody coupled to agarose were purchased fromSigma. Cell culture reagents were obtained from Life Sciences (Basel,Switzerland) and Biowhittaker (Walkersville, Md.). Flag-tagged solublehuman APRIL (residues K₁₁₀-L₂₅₀) was produced in 293 cells as described(10, 11). FITC-labeled anti-CD4, anti-CD8 and anti-CD19 antibodies werepurchased from Pharmingen (San Diego, Calif.). Goat F(ab)₂ specific forthe Fc₅, fragment of human IgM were purchased from JacksonImmunoResearch (West Grove, Pa.). Secondary antibodies were obtainedfrom either Pharmingen or from Jackson ImmunoResearch and used at therecommended dilutions.

Human embryonic kidney 293 T (12) cells and fibroblast cell lines(Table 1) were maintained in DMEM containing 10% heat-inactivated fetalcalf serum (FCS). Human embryonic kidney 293 cells were maintained inDMEM-nutrient mix F12 (1:1) supplemented with 2% FCS. T cell lines, Bcell lines, and macrophage cell lines (Table 1) were grown in RPMIsupplemented with 10% FCS. Molt-4 cells were cultiv-atedin Iscove'smedium-supplemented with 10% FCS. Epithelial cell lines were grown inMEM-alpha medium containing 10% FCS, 0.5 mM non-essential amino acids,10 mM Na-Hepes and 1 mM Na pyruvate. HUVECs were maintained in M199medium supplemented with 20% FCS, 100 μg/ml of epithelial cell growthfactor (Collaborative Research, Inotech, Dottikon, Switzerland) and 100μg/ml of heparin sodium salt (Sigma). All media contained penicillin andstreptomycin antibiotics. Peripheral blood leukocytes were isolated fromheparinized blood of healthy adult volunteers by Ficoll-Paque(Pharmacia, Uppsala, Sweden) gradient centrifugation and cultured inRPMI, 10% FCS.

T cells were obtained from non-adherents PBLs by rosetting withneuraminidase-treated sheep red blood cells and separated fromnon-rosetting cells (mostly B cells and monocytes) by Ficoll-Paquegradient centrifugation. Purified T cells were activated for 24 h withphytohemagglutinin (Sigma) (1 μg/ml), washed and cultured in RPMI, 10%FCS, 20 U/ml of IL 2. CD14⁺ monocytes were purified by magnetic cellsorting using anti-CD 14 antibodies, goat anti-mouse-coated microbeadsand a Minimacs™ device (Miltenyi Biotech), and cultivated in thepresence of GM-CSF (800 U/ml, Leucomax®, Essex Chemie, Luzern,Switzerland) and IL-4 (20 ng/ml, Lucerna Chem, Luzern, Switzerland) for5 d, then with GM-CSF, IL4 and TNFα(200 U/ml, Bender, Vienna, Austria)for an additional 3 d to obtain a CD83⁺, dentritic cell-like population.Human B cells of >97% purity were isolated from peripheral blood orumbilical cord blood using anti-CD19 magnetic beads (M450, Dynal, Oslo,Norway) as described (13).

Northern Blot Analysis

Northern blot analysis was carried out using Human Multiple TissueNorthern Blots I and II (Clontech #7760-1 and #7759-1). The membraneswere incubated in hybridization solution (50% formamide, 2.5×Denhardt's, 0.2% SDS, 10 mM EDTA, 2×SSC, 50 mM NaH₂PO₄, pH 6.5, 200μg/ml sonicated salmon sperm DNA) for 2 h at 60° C. Antisense RNA probecontaining the nucleotides corresponding to amino acids 136-285 of hBAFFwas heat-denatured and added at 2×10⁶ cpm/ml in fresh hybridizationsolution. The membrane was hybridized 16 h at 62° C., washed once in2×SSC, 0.05% SDS (30 min at 25° C.), once in 0.1×SSC, 0.1% SDS (20 minat 65° C.) and exposed at −70° C. to X-ray films.

Characterization of BAFF cDNA.

A partial sequence of human BAFF cDNA was contained in several ESTclones (e.g. GenBank Accession numbers T87299 and AA166695) derived fromfetal liver and spleen and ovarian cancer libraries. The 5′ portion ofthe cDNA was obtained by 5′-RACE-PCR (Marathon-Ready cDNA, Clonetech,Palo Alto, Calif.) amplification with oligonucleotides AP1 and JT1013(5′-ACTGTTTCTTCTGGACCCTGAACGGC-3′) [SEQ ID. NO.: 9] using the providedcDNA library from a pool of human leukocytes as template, as recommendedby the manufacturer. The resulting PCR product was cloned into PCR-0blunt (Invitrogen, Nev. Leek, The Netherlands) and subcloned asEcoRI/PstI fragment into pT7T3 Pac vector (Pharmacia) containing ESTclone T87299. Full-length hBAFF cDNA was therefore obtained by combining5′ and 3′ fragments using the internal PstI site of BAFF. Sequence hasbeen assigned GenBank accession number AF116456.

A partial 617 bp sequence of murine BAFF was contained in twooverlapping EST clones (AA422749 and AA254047). A PCR fragment spanningnucleotides 158 to 391 of this sequence was used as a probe to screen amouse spleen cDNA library (Stratagene, La Jolla, Calif.).

Expression of Recombinant BAFF

Full length hBAFF was amplified using oligos JT1069(5′-GACAAGCTTGCCACCATGGATGACTCCACA-3′) [SEQ. ID. NO.: 10] and JT637(5′-ACTAGTCACAGCAGTITCAATGC-3) [SEQ. ID. NO.: 11]. The PCR product wascloned into PCR-0 blunt and re-subcloned as HindIII/EcoRI fragment intoPCR-3 mammalian expression vector. A short version of soluble BAFF(amino acids Q136-L285) was amplified using oligos JT636(5′-CTGCAGGGTCCAGAAGAAACAG-3) [SEQ. ID. NO.: 12] and JT637. A longversion of soluble BAFF (aa L83-L285) was obtained from full length BAFFusing internal PstI site. Soluble BAFFs were resubcloned as PstI/EcoRIfragments behind the haemaglutinin signal peptide and Flag sequence of amodified PCR-3 vector, and as PstI/SpeI fragments into a modified pQE16bacterial expression vector in frame with a N-terminal Flag sequence(14). Constructs were sequenced on both strands. The establishment ofstable 293 cell lines expressing the short soluble form or full lengthBAFF, and the expression and purification of recombinant soluble BAFFfrom bacteria and mammalian 293 cells was performed as described (14,15).

Reverse Transcriptase PCR

Total RNA extracted from T cells, B cells, in vitro derived immaturedendritic cells, 293 wt and 293-BAFF (full length) cells was reversetranscribed using the Ready to Go system (Pharmacia) according to themanufacturer's instructions. BAFF and β-actin cDNAs were detected by PCRamplification with Taq DNA polymerase (steps of 1 min each at 94° C.,55° C. and 72° C. for 30 cycles) using specific oligonucleotides: forBAFF, JT1322 5′-GGAGAAGGCAACTCCAGTCAGAAC-3′ [SEQ. ID. NO.: 13] andJT1323 5′-CAATTCATCCCCAAAGACATGGAC-3′ [SEQ. ID. NO.: 14]; for IL-2receptor alpha chain, JT1368 5′-TCGGAACACAACGAAACAAGTC-3′ [SEQ. ID. NO.:15] and JT1369 5′-CTTCTCCTTCACCTGGAAACTGACTG-3′ [SEQ. ID NO.: 16]; forβ-actin, 5′-GGCATCGTGATGGACTCCG-3′ [SEQ. ID. NO.: 17] and5′-GCTGGAAGGTGGACAGCGA-3′ [SEQ. ID. NO.: 18].

Gel Permeation Chromatography

293T cells were transiently transfected with the short form of solubleBAFF and grown in serum-free Optimem medium for 7 d. Conditionnedsupernatants were concentrated 20×, mixed with internal standardscatalase and ovalbumin, and loaded onto a Superdex-200 HR10/30 column.Proteins were eluted in PBS at 0.5 ml/min and fractions (0.25 ml) wereprecipitated with trichloroacetic acid and analyzed by Western blottingusing anti-Flag M2 antibody. The column was calibrated with standardproteins: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa),bovine serum albumine (67 kDa), ovalbumine (43 kDa), chymotrypsinogen A(25 kDa) and ribonuclease A (13.7 kDa).

PNGase F Treatment

Samples were heated in 20 μl of 0.5% SDS, 1% 2-mercaptoethanol for 3 minat 95° C., then cooled and supplemented with 10% Nonidet P40 (2 μl), 0.5M sodium phosphate, pH 7.5 (2 μl) and Peptide N-glycanase F (125units/μl, 1 μl, or no enzyme in controls). Samples were incubated for 3h at 37° C. prior to analysis by Western blotting.

EDMAN Sequencing

293 T cells were transiently transfected with the long form of solubleBAFF and grown in serum-free Optimem medium for 7 d. Conditionedsupernatants were concentrated 20×, fractionated by SDS-PAGE and blottedonto polyvinylidene difluoride membrane (BioRad Labs, Hercules, Calif.)as previously described (16), and then sequenced using a gas phasesequencer (ABI 120A, Perkin Elmer, Foster City, Calif.) coupled to ananalyzer (ABI 120A, Perkin Elmer) equipped with a phenylthiohydantoinC18 2.1×250 mm column. Data was analyzed using software ABI 610 (Perkin.Elmer).

Antibodies

Polyclonal antibodies were generated by immunizing rabbits (Eurogentec,Seraing, Belgium) with recombinant soluble BAFF. Spleen of ratsimmunized with the same antigen were fused to x63Ag8.653 mouse myelomacells, and hybridoma were screened for BAFF-specific IgGs. One of thesemonoclonal antibodies, 43.9, is an IgG2a that specifically recognizeshBAFF.

Cells were stained in 50 μl of FACS buffer (PBS, 10% FCS, 0.02% NaN₃)with 50 ng (or the indicated amount) of Flag tagged short soluble HBAFFfor 20 min at 4° C., followed by anti-Flag M2 (1 μg) and secondaryantibody. Anti-BAFF mAb 43.9 was used at 40 μg/ml. For two color FACSanalysis, peripheral blood lymphocytes were stained with Flag taggedsoluble BAFF/long (2 μg/ml), followed by biotinylated anti-Flag M2(1/400) and PE-labeled streptavidin (1/100), followed by eitherFITC-labeled anti-CD4, anti-CD8 or anti-CD19.

PBL Proliferation Assay

Peripheral blood leukocytes were incubated in 96-well plates (10⁵cells/well in 100 μl RPMI supplemented with 10% FCS) for 72 h in thepresence or absence of 2 μg/ml of goat anti-human p chain antibody(Sigma) or control F(ab′)₂ and with the indicated concentration ofnative or boiled soluble BAFF/long. Cells were pulsed for an additional6 h with [³H]thymidine (1 μCi/well) and harvested. [³H]thymidineincorporation was monitored by liquid scintillation counting. In someexperiments, recombinant soluble BAFF was replaced by 293 cells stablytransfected with full length BAFF (or 293 wt as control) that had beenfixed for 5 min at 25° C. in 1% paraformaldeyde. Assay was performed asdescribed (17). In further experiments, CD19⁺ cells were isolated formPBL with magnetic beads and the remaining CD19⁻ cells were irradiated(3000 rads) prior to renconstitution with CD19+cells. Proliferationassay with sBAFF was then performed as described above.

B cell Activation Assay

Purified B cells were activated in the EL4 culture system as described(13). Briefly, 10⁴ B cells mixed with 5×10⁴ irradiated murine ELAthymoma cells (clone B5) were cultured for 5-6 d in 200 μl mediumcontaining 5% v/v of culture supernatants from human T cells (106/ml)which had been activated for 48 h with PHA (1 μg/ml) and PMA (1 ng/ml).B cells were then reisolated with anti-CD19 beads and cultured foranother 7 d (5×10⁴ cells in 200 μl, duplicate or triplicate culture inflat bottomed 96 well plates) in medium alone or in medium supplementedwith 5% T cell supernatants, or with 50 ng/ml IL-2 (a kind gift from theformer Glaxo Institute for Molecular Biology, Geneva) and 10 ng/ml eachIL4 and IL-10 (Peprotech, London, UK), in the presence or absence ofsBAFF. The anti-Flag M2 antibody was added at a concentration of 2 μg/mland had no effect by itself. IgM, IgG and IgA in culture supernatantswere quantitated by ELISA assays as described (13).

Human BAFF was identified by sequence homology as a possible novelmember of the TNF ligand family while we screened public databases usingan improved profile search (18). A cDNA encoding the complete protein of285 amino acids (aa) was obtained by combining EST-clones (covering the3′ region) with a fragment (5′ region) amplified by PCR. The absence ofa signal peptide suggested that BAFF was a type II membrane protein thatis typical of the members of the TNF-ligand family. The protein has apredicted cytoplasmic domain of 46 aa, a hydrophobic transmembraneregion, and an extracellular domain of 218 aa containing two potentialN-glycosylation sites (FIG. 1A). The sequence of the extracellulardomain of BAFF shows highest homology with APRIL (33% amino acididentities, 48% homology), whereas the identity with other members ofthe family such as TNF, FasL, LTa, TRAIL or RANKL is below 20% (FIG. 1B,C). The mouse BAFF cDNA clone isolated from a spleen library encoded aslightly longer protein (309 aa) due to an insertion between thetransmembrane region and the first of several β-strands which constitutethe receptor binding domain in all TNF ligand members (19). Thisβ-strand rich ectodomain is almost identical in mouse and human BAFF(86% identity, 93% homology) suggesting that the BAFF gene has beenhighly conserved during evolution (FIG. 1A).

Although TNF family members are synthesized as membrane-insertedligands, cleavage in the stalk region between transmembrane and receptorbinding domain is frequently observed. For example, TNF or FasL arereadily cleaved from the cell surface by metalloproteinases (20, 21).While producing several forms of recombinant BAFF in 293T cells, wenoticed that a recombinant soluble 32 kDa form of BAFF (aa 83-285,sBAFF/long), containing the complete stalk region and a N-terminalFlag-tag in addition to the receptor binding domain, was extensivelyprocessed to a smaller 18 kDa fragment (FIG. 2A, B). Cleavage occurredin the stalk region since the fragment was detectable only withantibodies raised against the complete receptor interaction domain ofBAFF but not with anti-Flag antibodies (data not shown). Also revealedwas that only N124 (located in the stalk) but not N242 (located at theentry of the F-□ sheet) was glycosylated, since the molecular mass ofthe non-processed sBAFF/long was reduced from 32 kDa to 30 kDa uponremoval of the N-linked carbohydrates with PNGase F whereas the 18 kDacleaved form was insensitive to this treatment. Peptide sequenceanalysis of the 18 kDa fragment indeed showed that cleavage occurredbetween R133 and A134 (FIG. 1A). R133 lies at the end of a polybasicregion which is conserved between human (R-N-K-R) and mouse (R-N-R-R).To test whether cleavage was not merely an artifact of expressingsoluble, non-natural forms of BAFF, membrane-bound full length BAFF wasexpressed in 293T cells (FIG. 2C). The 32 kDa complete BAFF and somehigher molecular mass species (probably corresponding to non-dissociateddimers and trimers) were readily detectable in cellular extracts, butmore than 95% of BAFF recovered from the supernatant corresponded to theprocessed 18 kDa form, indicating that BAFF was also processed whensynthesized as a membrane-bound ligand.

A soluble BAFF was engineered (Q136-L285, sBAFF/short) whose sequencestarted 2 aa downstream of the processing site (FIG. 1 B). As predicted,the Flag-tag attached to the N-terminus of this recombinant molecule wasnot removed (data not shown) which allowed its purification by ananti-Flag affinity column. To test its correct folding, the purifiedsBAFF/short was analyzed by gel filtration where the protein eluted atan apparent molecular mass of 55 kDa (FIG. 2D). The sBAFF/shortcorrectly assembles into a homotrimer (3×20 kDa) in agreement with thequaternary structure of other TNF family members (19). Finally,unprocessed sBAFF/long was readily expressed in bacteria, indicatingthat the cleavage event was specific to eukaryotic cells.

Northern blot analysis of BAFF revealed that the 2.5 kb BAFF mRNA wasabundant in the spleen and PBLs (FIG. 3A). Thymus, heart, placenta,small intestine and lung showed weak expression. This restricteddistribution suggested that cells present in lymphoid tissues were themain source of BAFF. Through PCR analysis, we found that BAFF mRNA waspresent in T cells and peripheral blood monocyte-derived dendritic cellsbut not in B cells (FIG. 3B). Even naive, non-stimulated T cellsappeared to express some BAFF mRNA.

A sequence tagged site (STS, SHGC-36171) was found in the database whichincluded the human BAFF sequence. This site maps to human chromosome 13,in a 9 cM interval between the markers D13S286 und D13S1315. On thecytogenetic map, this interval corresponds to 13q32-34. Of the known TNFligand family members, only RANKL (Trance) has been localized to thischromosome (22) though quite distant to BAFF (13q14).

In order for the ligand to exert maximal biological effects, it waslikely that the BAFF receptor (BAFF-R) would be expressed either on thesame cells or on neighboring cells present in lymphoid tissues. Usingthe recombinant sBAFF as a tool to specifically determine BAFF-Rexpression by FACS, we indeed found high levels of receptor expressionin various B cell lines such as the Burkitt lymphomas Raji and BJAB(FIG. 4A, Table 1). In contrast, cell lines of T cell, fibroblastic,epithelial and endothelial origin were all negative. Very weak stainingwas observed with the monocyte line THP-1 which, however, could be dueto Fc receptor binding. Thus, BAFF-R expression appears to be restrictedto B cell lines. The two mouse B cell lines tested were negative usingthe human BAFF as a probe, although weak binding was observed on mousesplenocytes (data not shown). The presence of BAFF-R on B cells wascorroborated by analysis of umbilical cord and peripheral bloodlymphocytes. While CD8⁺ and CD4⁺ T cells lacked BAFF-R (FIG. 4B and datanot shown), abundant staining was observed on CD19⁺ B cells (FIGS. 4Aand 4B), indicating that BAFF-R is expressed on all blood B cells,including naive and memory ones.

Since BAFF bound to blood-derived B cells, experiments were performed todetermine whether the ligand could deliver growth-stimulatory or-inhibitory signals. Peripheral blood lymphocytes (PBL) were stimulatedwith anti-IgM (μ) antibodies together with fixed 293 cells stablyexpressing surface BAFF (FIG. 5A). The levels of [³H]thymidineincorporation induced by anti-μ alone was not altered by the presence ofcontrol cells but was increased two-fold in the presence ofBAFF-transfected cells (FIG. 5B). A dose-dependent proliferation of PBLwas also obtained when BAFF-transfected cells were replaced by purifiedsBAFF (FIG. 5C), indicating that BAFF does not require membraneattachment to exert its activity. In this experimental setup,proliferation induced by sCD40L required concentrations exceeding 1μg/ml but was less dependent on the presence of anti-μ than thatmediated by BAFF (FIG. 5D). When purified CD19⁺ B cells were co-culturedwith irradiated autologous CD19⁻ PBL, costimulation of proliferation byBAFF was unaffected, demonstrating that [³H]thymidine uptake was mainlydue to B cell proliferation and not to an indirect stimulation ofanother cell type (data not shown). The observed B cell proliferation inresponse to BAFF was entirely dependent on the presence of anti-μantibodies, indicating that BAFF functioned as costimulator of B cellproliferation.

To investigate a possible effect of BAFF on immunoglobulin secretion,purified peripheral or cord blood B cells were preactivated by coculturewith EL-4 T cells in the presence of a cytokine mixture fromsupernatants of PHA/PMA stimulated T cells (23). These B cells werereisolated to 98% purity and yielded a two-fold increase in Ig secretionduring a secondary culture in the presence of BAFF and activated T cellcytokines as compared to cytokines alone. A very modest effect occurredin the absence of exogenous cytokines, and an intermediate (1.5-fold)effect was observed in the presence of the recombinant cytokines IL-2,IL4 and IL-10 (FIG. 5E, F).

The biochemical analysis of BAFF is also consistent with the typicalhomotrimeric structure of TNF family members. Among this family ofligands, BAFF exhibits the highest level of sequence similarity withAPRIL which we have recently characterized as a ligand stimulatinggrowth of various tumor cells (11). Unlike TNF and LTO which are twofamily members with equally high homology (33% identity) and whose genesare linked on chromosome 6, APRIL and BAFF are not clustered on the samechromosome. APRIL is located on chromosome 17 (J. L. B., unpublisheddata) whereas BAFF maps to the distal arm of human chromosome 13(13q34). Abnormalities in this locus were characterized in Burkittlymphomas as the second most frequent defect (24) besides thetranslocation involving the myc gene into the Ig locus (25). Consideringthe high expression levels of BAFF-R on all Burkitt lymphoma cell linesanalyzed (see Table 1), this raises the intriguing possibility that someBurkitt lymphomas may have deregulated BAFF expression, thus stimulatinggrowth in an autocrine manner.

The role of antigen-specific B lymphocytes during the different stagesof the immune response is highly dependent on signals and contacts fromhelper T cells and antigen-presenting cells such as dendritic cells(20). B lymphocytes first receive these signals early on during theimmune response when they interact with T cells at the edge of the Bcell follicles in lymphoid tissues, leading to their proliferation anddifferentiation into low affinity antibody forming cells (18). At thesame time some antigen-specific B cells also migrate to the B cellfollicle and contribute to the formation of germinal centers, anothersite of B cell proliferation but also affinity maturation and generationof memory B cells and high affinity plasma cells (19).

Signals triggered by another member of the TNF super family CD40L havebeen shown to be critical for the function of B lymphocytes at multiplesteps of the T cell-dependent immune response. However, several studiesclearly showed that CD40UCD40 interaction does not account for allcontact-dependent T-cell help for B cells. Indeed, CD40L-deficient Tcells isolated from either knock-out mice or patients with X-linkedhyper IgM syndrome have been shown to sucessfully induce proliferationof B cells and their differentiation into plasma cells. Studies usingblocking antibodies against CD40L showed that a subset of surface IgDpositive B cells isolated from human tonsils proliferate anddifferentiate in response to activated T cells in a CD40-independentmanner. Other members of the TNF family such as membrane-bound TNF andCD30L have also been shown to be involved in a CD40- and surfaceIg-independent stimulation of B cells. Similar to our results with BAFF,it has been shown that CD40-deficient B cells can be stimulated toproliferate and differentiate into plasma cells by helper T cells aslong as the surface Ig receptors are triggered at the same time. BAFF aswell as CD30L and CD40L is expressed by T cells but its originalityresides in its expression by dendritic cells as well as the highlyspecific location of its receptor on B cells which is in contrast toCD40, CD30 and the TNF receptor which expression has been descrided onmany different cell. This observation suggests independent and specificBAFF-induced functions on B cells.

In support of a role for BAFF in T cell- and dendritic cell-induced Bcell growth and potential maturation, we found that BAFF costimulatesproliferation of blood-derived B cells concomitantly with cross-linkingof the B cell receptors, and, thus, independently of CD40 signalling.Moreover, using CD19 positive B cells differentiated in vitro into apre-plasma cell/GC-like B cell (14), we observed a costimulatory effectof BAFF on Ig secretion by these B cells in the presence of supernatantfrom activated T cells or a blend of IL-2, IL-4 and IL-10.Interestingly, the costimulatory effect was stronger in presence of theactivated T cell supernatant when compared to the cytokine blend,suggesting additional soluble factors secreted by activated T cellsinvolved in antibody production which can synergize with BAFF oradditional BAFF itself. It is, therefore, possible that BAFF activelycontributes to the differentiation of these GC-like B cells into plasma.

It is clear that BAFF can signal in both naive B cells as well asGC-conunited B cells in vitro. Whether this observation will translateor not during a normal immune response will have to be addressed byproper in vivo experiments.

The biological responses induced in B cells by BAFF are distinct fromthat of CD40L, since proliferation triggered by CD40L was less dependenton an anti-μ costimulus (17) (and FIG. 5D). Morever, CD40L cancounteract apoptotic signals in B cells following engagement of the Bcell receptor (29), whereas BAFF was not able to rescue the B cell lineRamos from anti-μ-mediated apoptosis, despite the fact that Ramos cellsdo express BAFF-R (Table 1; F. M. and J. L. B., unpublishedobservations). It is therefore likely that CD40L and BAFF fulfilldistinct functions. In this respect, it is noteworthy that BAFF did notinteract with any of 16 recombinant receptors of the TNF family tested,including CD40 (P.S and J.T, unpublished observations).

B cell growth was efficiently costimulated with recombinant soluble BAFFlacking the transmembrane domain. This activity is in contrast toseveral TNF family members which are active only as membrane-boundligand such as TRAIL, FasL and CD40L. Soluble forms of these ligandshave poor biological activity which can be enhanced by theircross-linking, thereby mimicking the membrane-bound ligand (15). Incontrast, cross-linking Flag-tagged sBAFF with anti-Flag antibodies orthe use of membrane-bound BAFF expressed on the surface of epithelialcells did not further enhance the mitogenic activity of BAFF, suggestingthat it can act systemically as a secreted cytokine, like TNF does. Thisis in agreement with the observation that a polybasic sequence presentin the stalk of BAFF acted as a substrate for a protease. Similarpolybasic sequences are also present at corresponding locations in bothAPRIL and TWEAK and for both of them there is evidence of proteolyticprocessing (30) (N.H. and J.T, unpublished observation). Although theprotease responsible for the cleavage remains to be determined, it isunlikely to be the metalloproteinase responsible for the release ofmembrane-bound TNF as their sequence preferences differ completely (21).The multibasic motifs in BAFF (R-N-K-R), APRIL (R-K-R-R) and Tweak(R-P-R-R) are reminiscent of the minimal cleavage signal for furin(R-X-K/R-R), the prototype of a proprotein convertase family (31).

Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. See, for example, Molecular Cloning: ALaboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.),Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II(D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.),1984; U.S. Pat. No. 4,683,195 (Mullis et al.,); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcriptionand Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture ofAnimal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; ImmobilizedCells and Enzymes, IRL Press, 1986; A Practical Guide to MolecularCloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155(Wu et al., eds), Academic Press, New York; Gene Transfer Vectors forMammalian Cells (J. H. Miller and M. P. Calos, eds.), 1987, Cold SpringHarbor Laboratory; Immunochemical Methods in Cell and Molecular Biology(Mayer and Walker, eds.), Academic Press, London, 1987; Handbook ofExperiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds.), 1986; Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, 1986.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLES

The following experimental procedures were utilized in Examples 1-6.

DNA Construct for the Generation of Murine BAFF Tg Mice

Both human and murine cDNA sequences have been described previously(Schneider et al., 1999). A PCR fragment encoding full-length murineBAFF was generated by RT-PCR. First strand cDNA was synthesized frommouse lung polyA+ (Clontech, Palo Alto, Calif.) using oligo dT accordingto the manufacturer's protocol (GibcoBRL, (Grand Island, N.Y.). The PCRreaction contained 1×Pfu buffer (Stratagene, La Jola, Calif.), 0.2 mMdNTPs, 10% DMSO, 12.5 pM primers, 5 units Pfu enzyme (Stratagene) andthe following primers with Not1 restriction sites5′-TAAGAATGCGGCCGCGGAATGGATGAGTCTGCAAA-3′ [SEQ. ID. NO.: 19] and5′-TAAGAATGCGGCCGCGGGATCACGCACTCCAGCAA-3′ [SEQ. ID. NO.: 20]. Thetemplate was amplified for 30 cycles at 94° C. for 1 min, 54° C. for 2min and 72° C. for 3 min followed by a 10 min extension at 72° C. Thissequence corresponds to nucleotides 214 to 1171 of the GenBank fileAF119383. The PCR fragment was digested with Not1 and then cloned into amodified pCEP4 vector (Invitrogen, Carlsbad, Calif.). The fragmentcontaining murine BAFF was removed with Xba1 in order to include theSV40 polyA addition site sequence. This fragment was cloned into a pUCbased vector where the promoter sequence was added. The promoter, a 1 Kbblunt Bgl2-Not1 fragment containing the human ApoE enhancer and AAT(alpha anti-trypsin) promoter was purified from the plasmid clone 540B(a kind gift from Dr. Katherine Parker Ponder, Washington University,St. Louis, Mo.). An EcoRV/Bgl2 fragment was purified from the finalvector and used for the generation of transgenic mice. The injectedoffspring of C57BL/6J female×DBA/2J male F1 (BDF1) mice were backcrossedonto C57BL/6 mice. Techniques of microinjection and generation oftransgenic mice have been previously described (Mcknights et al., 1983).

Analytical Methods:

Serum samples were subject to reduced SDS-PAGE analysis using a linear12.5% gel. Total RNA from mouse liver was prepared and processed forNorthern Blot analysis using an isolation kit from Promega (Madison,Wis.) according to the manufacturer's guidelines. BAFFtransgene-specific mRNA was detected using a probe spanning the SV40poly A tail of the transgene construct and obtained by digestion of themodified pCEP4 vector with Xba1 and BamH1. The probe recognizes a 1.8-2Kd band corresponding to mRNA from the BAFF transgene. PCR analysis oftail DNA from BAFF Tg mice was carried using 12.5 pM of the followingprimers 5′-GCAGTTTCACAGCGATGTCCT-3′ [SEQ. ID. NO.: 21] and5′-GTCTCCGTTGCGTGAAATCTG-3′ [SEQ. ID. NO.: 22] in a reaction containing1× Taq polymerase buffer (Strata gene), 0.2 nM dNTPs, 10% DMSO and 5units of Taq polymerase (Stratagene). A 719 bp of the transgene wasamplified for 35 cycles at 94° C. for 30 sec., 54° C. for 1 min. and 72°C. for 1.5 min. followed by a 10 min. extension at 72° C.

The presence of proteins in mouse urine was measured using Multistix 10SG reagent strips for urinalysis (Bayer Corporation, DiagnosticsDivision, Elkhart, Ind.).

Cell-dyn and Cytofluorimetric Analysis (FACS).

Differential WBC counts of fresh EDTA anticoagulated whole blood wereperformed with an Abbott Cell Dyne 3500 apparatus (Chicago, Ill.). ForFACS analysis, Fluorescein (FITC)-, Cy-chrome- andPhycoerythrin-(PE)-labeled rat anti-mouse antibodies: anti-B220,anti-CD4, anti-CD8, anti-CD43, anti-IgM, anti-CD5, anti-CD25, anti-CD24,anti-CD38, anti-CD21, anti-CD44, anti-L-selectin and hamsteranti-Bcl-2/control hamster Ig kit were purchased from Pharmingen (SanDiego, Calif.). Production of recombinant E. coli as well as mammaliancell-derived human and mouse Flag-tagged BAFF were previously described(Schneider et al., 1999). All antibodies were used according to themanufacturer's specifications. PBL were purified from mouse blood asfollows: mouse blood was collected in microtubes containing EDTA and wasdiluted ½ with PBS. Five hundred μl of diluted blood was applied on topof 1 ml of ficoll (Celardane, Hornby, Ontario, Canada) in a 4 ml glasstube, the gradient was performed at 2000 rpm for 30 min at roomtemperature and the interface containing the lymphocytes was collectedand washed twice in PBS prior to FACS staining. Spleen, bone marrow andmesenteric lymph nodes were ground into a single cell suspension in RPMImedium (Life Technologies, Inc., Grand Island, N.Y.) and washed in FACSbuffer (PBS supplemented with 2% fetal calf serum (JRH Biosciences,Lenexa, Kans.). Cells were first suspended in FACS buffer supplementedwith the following blocking reagents: 10 μg/ml human Ig (Sandoz, Basel,Switzerland) and 10 μg/ml anti-mouse Fc blocking antibody (Pharmingen)and incubated 30 min on ice prior to staining with fluorochrome-labeledantibodies. All antibodies were diluted in FACS buffer with the blockingreagent mentioned above. Samples were analyzed using a FACScancytofluorometer (Becton Dickinson).

Detection of Total Mouse Ig and Rheumatoid Factors in Mouse Sera byELISA Assays.

ELISA plates (Corning glass works, Corning, N.Y.) were coated overnightat 4° C. with a solution of 10 μg/ml of goat anti-total mouse Ig(Southern Biotechnology Associates, Inc. Birmingham, Ala.) in 50 mMsodium bicarbonate buffer pH 9.6. Plates were washed 3 times withPBS/0.1% Tween and blocked overnight with 1% gelatin in PBS. One hundredμl/well of serum serial dilutions or standard dilutions was added to theplates for 30 min at 37° C. Mouse Ig were detected using 100 μl/well ofa 1 μg/ml solution of an Alkaline Phosphatase (AP)-labeled goatanti-total mouse Ig (Southern Biotechnology Associates) for 30 min at37° C. After a last wash, 3 times with PBS/0.1% Tween, the enzymaticreaction was developed using a solution of 10 μg/ml of p-nitrophenylphosphate (Boehringer Mannheim, Indianapolis, Ind.) in 10%diethanolamine. The reaction was stopped by adding 100 μl of 3NNaOH/well. The optical density (O.D.) was measured at 405 nm using aspectrophotometer from Molecular Devices (Sunnyvale, Calif.). Standardcurves were obtained using purified mouse Ig purchased from SouthernBiotechnology Associates. In the case of detection of rheumatoid factors(RF), the plates were coated with normal goat Ig (Jackson ImmunoResearchlaboratories, Inc., West Grove, Pa.) instead of goat anti-mouse Ig anddetection of mouse Ig was performed as described above. Detection ofmouse isotypes in the RF assay was done using AP-labeled goat anti-mouseIgA, IgM, IgG2a, IgG2b and IgG3, as well as purified mouse IgA, IgM,IgG2a, IgG2b and IgG3 for standard curves (Southern BiotechnologyAssociates Inc.). All statistical comparisons were performed by analysisof variance.

Detection of Circulating Immune Complexes (CIC) and Precipitation ofCryoglobulins in Mouse Sera.

The assay was performed as previously described (June et al., 1979;Singh and Tingle, 1982) with the following modifications: ELISA plates(Corning glass works) were coated overnight at 4° C. with 5 μg/ml ofhuman C1q (Quidel, San Diego, Calif.) in 50 mM sodium bicarbonate bufferpH 9.6. The plates were washed 3 times with PBS/0.1% Tween. Fiftyμl/well of 0.3 M EDTA was added to the plates plus 50 μl/well of serumserial dilutions or solutions of known concentrations of a standardimmune complex (peroxidase-mouse anti-peroxidase (PAP) from DAKO(Carpinteria, Calif.). The plates were incubated 30 min at 37° C. Theplates were washed 3 times with PBS/0.1% Tween. Mouse Ig in the immunecomplexes were detected using 100 μl/well of a 1 μg/ml solution of anAP-labeled goat anti-mouse Ig (Southern Biotechnology Associates, Inc.)as described above for the ELISA assays. Cryoglobulins were detected byincubating overnight at 4° C. mouse serum diluted 1/15 in water andprecipitates were scored visually.

Anti-Double Stranded (ds) and Single Stranded (ss) DNA Assays.

Anti-ssDNA were performed using NUNC-immuno Plate MaxiSorp plates (NUNCA/S, Denmark). Plates were coated overnight at 4° C. first with 100μg/ml methylated BSA (Calbochem Corp., La Jolla, Calif.), then with 50μg/ml grade I calf thymus DNA (Sigma, St. Louis, Mo.). The calf thymusDNA was sheared by sonication and then digested with S1 nuclease beforeuse. For the anti-ssDNA assay, the DNA was boiled for 10 min and chilledon ice before use. After blocking, serial dilutions of the serum sampleswere added and incubated at room temperature for 2 h. Autoantibodieswere detected with goat anti-mouse IgG-AP (Sigma) and develop asdescribed above for the ELISA assays. Standard curves were obtainedusing known quantities of anti-DNA mAb 205, which is specific for bothss- and dsDNA (Datta et al., 1987).

Immunohistochemistry

Spleen and lymph nodes were frozen in O.C.T. embedding medium (Miles,Elkhart, Ind.) and mounted for cryostat sectioning. Sections 7-10 μmthick were dried and fixed in acetone. All Ab incubations (10 μg/ml)were done for 1 hr at room temperature in a humidified box afterdilution in Tris-buffered saline A (TBS-A, 0.05M Tris, 0.15M NaCl, 0.05%Tween-20 (v/v), 0.25% BSA), rinsed in TBS-B (0.05M Tris, 0.15M NaCl,0.05% Tween-20) and fixed 1 min in methanol before initiating theenzymatic reaction. Horseradish peroxidase (HRP) and alkalinephosphatase (AP) activities were developed using the diaminobenzidine(DAB) tablet substrate kit (Sigma) and the 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT, Pierce, Rockford, Ill.),respectively. Stained tissue sections were finally fixed 5 min inmethanol and counter stained with Giemsa (Fluka, Buchs, Switzerland).Biotin-labeled antibodies rat anti-B220, anti-CD11c, anti-syndecan-1 aswell as unlabeled rat anti-CD4, anti-CD8α and anti-CD8β were purchasedfrom Pharmingen. Biotin-labeled peanut agglutinin (PNA) was obtainedfrom Vector laboratories (Burlingame, Calif.). (HRP)-labeled mouseanti-rat Ig and (HRP)-streptavidin were purchased from JacksonImmunoResearch laboratories, Inc. and AP-labeled streptavidin fromSouthern Biotechnology Associates, Inc. In the case ofimmunohistochemistry on kidney tissue to detect Ig deposition, paraffinsection were used, dewaxed and blocked using diluted horse serum fromVector (Burlingame, Calif.), followed by staining with HRP-goatanti-mouse Ig from Jackson Immunoresearch. Detection was performed asdescribed above.

Example 1

BAFF Transgenic (BAFF Tg) Founder Mice have an Abnormal Phenotype.

Full length murine BAFF was expressed in transgenic mice using the liverspecific alpha-1 antitrypsin promoter with the APO E enhancer. The fulllength version was chosen with the expectation that BAFF would be eithercleaved and act systemically or if retained in a membrane bound formthat local liver specific abnormalities would be observed possiblyproviding functional clues. We obtained 13 founder mice positive for theBAFF transgene (Table 2). Four of these mice died at a young age.Routine pathology was carried out on mice 811 and 816 (Table 2). Therewas no obvious infection in these mice; however, cardiovascular andrenal abnormalities were apparent and similar to those described forsevere hypertension (Fu, 1995) (Table 2). Hematoxylin and eosin(H&E)-stained kidney tissue sections of founder 816 showed that themorphology of glomeruli in that mouse was abnormal, whereas the rest ofthe kidney tissue seemed normal (data not shown). Many BAFF transgenicfounder mice had proteinuria (Table 2). Immunohistochemistry on spleenfrozen tissue sections from mouse 816, revealed an abnormal andextensive B cell staining and reduced staining for T cells and thisobservation was confirmed in the progeny (see below, FIG. 12).

Using two color FACS analysis, the ratio of % B220 positive B cells over% CD4 positive T cells was calculated. This ratio was two to seven timeshigher in BAFF Tg founder mice when compared to control negative BDF1mice (Table 2), suggesting an increase of the B cell population in BAFFTg mice. We selected nine of these founder mice to generate ourdifferent lines of transgenic mice as underlined in Table 2. None of theremaining BAFF Tg founder mice or the derived progeny showed any signsof ill health months after the early death of founders 696, 700, 811 and816, suggesting that these 4 mice might have expressed higher levels ofBAFF which caused their death. BAFF overexpression in the liver oftransgenic mice was confirmed by Northern blot analysis (data notshown). In all BAFF-Tg mice examined histologically, the livers showedno abnormalities indicating that local overexpression of BAFF did notinduce any immunological or pathological events. An ELISA assay formurine BAFF is not available; however, we showed that 2% serum from BAFFTg mice, but not from control mice, blocked the binding of mammaliancell-derived mouse soluble Flag-tagged BAFF to BJAB cells. Moreover, 5%serum from BAFF Tg mice but not from control mice increased theproliferation of human B cells from PBL in the presence of anti-μ (datanot shown). These data suggest that substantial amounts of soluble BAFFare present in the blood of BAFF Tg.

Example 2

Peripheral Lymphocytosis in BAFF Tg Mice is Due to Elevated B CellNumbers

The transgenic mice population was found to have more lymphocytes in theblood when compared to control negative littermates, reaching values ashigh as 13000 lymphocytes/μl of blood (FIG. 7A). In contrast, the numberof granulocytes per μl of blood in both BAFF Tg mice and control miceremained within normal limits (FIG. 7A). Since FACS analysis, usinganti-CD4 and anti-B220 antibodies, of peripheral blood cells (PBL) from18 BAFF Tg mice issued from six different founder mice showed increasedB/T ratios (FIGS. 7B and 7C), the elevated lymphocyte levels resultedfrom an expanded B cell subset. Likewise, using this method, calculationof absolute numbers of CD4 circulating T cells revealed a 50% reductionof this T cell subset in BAFF Tg mice when compared to control mice, andthe same observation was made for the CD8 T cell subset (data notshown). All B cells from the PBL of BAFF Tg mice have increased MHCclass II and Bcl-2 expression when compared to B cells from control mice(FIGS. 7D and 7E, respectively), indicating some level of B cellactivation in PBL of BAFF Tg mice. T cells in the blood of BAFF Tg micedid not express the early activation markers CD69 or CD25; however, 40to 56% of CD4 or CD8 T cells were activated effector T cells with aCD44^(hi), L-selectin^(lo) phenotype versus only 8% to 12% in controllittermates (FIG. 7F). Thus BAFF Tg mice clearly show signs of B celllymphocytosis and global B cell activation along with T cellalterations.

Example 3

Expanded B Cell Compartments are Composed of Mature Cells.

To see whether overexpression of BAFF in the transgenic mice wasaffecting the B cell compartment centrally in the bone marrow andperipherally in secondary lymphoid organs, we examined by FACS thespleen, bone marrow and mesenteric lymph nodes from a total of sevenBAFF Tg mice and seven control littermates derived from four differentfounder mice. The mature B cell compartment was analyzed by stainingwith both anti-B220 and anti-IgM antibodies. Two representative BAFF Tgmice and one representative control littermate are shown in FIG. 8. Themature B cell compartment (IgM+. B220+) was increased in both the spleenand the mesenteric lymph nodes (FIG. 8A, top and bottom panels,respectively). Analysis of B220+/IgM+B cells (FIG. 7A, middle panel) orthe proB cell (CD43+/B220+) and the preB cell (CD43−/B220+) compartmentsin the bone marrow (FIG. 8B) showed that BAFF Tg mice and controllittermates were similar. These data indicate that overexpression ofBAFF is affecting the proliferation of mature B cells in the peripherybut not progenitor B cells in the bone marrow. Analysis by FACS of the Bcell subpopulations in the spleen, revealed an increased proportion ofmarginal zone (MZ) B cells in BAFF Tg mice when compared to control mice(Table 3). The population of follicular B cells remained proportional inboth BAFF Tg and control mice whereas the fraction of newly formed Bcells is slightly decreased in BAFF Tg mice (Table 3). This result wasalso confirmed on B220+splenic B cells using anti-CD38 versus anti-CD24antibodies and anti-IgM versus anti-IgD antibodies and analyzing for atthe CD38^(hi)/CD24⁺ and IgM^(hi)/IgD^(lo) for the MZ B cell population,respectively, as previously described (Oliver et al., 1997)(data notshown). Immunohistochemical analysis using an anti-mouse IgM antibodyrevealed the expansion of the IgM-bright MZ B cell area in the spleen ofBAFF Tg mice when compared to control mice (data not shown). All BAFF TgB220+splenic B cells also express higher levels of MHC class II (Table3) and Bcl-2 (data not shown) compared to splenic B cells from controlmice, indicating that splenic B cells as well as B cells from PBL are inan activated state.

Example 4

BAFF Tg Mice have High Levels of Total Immunoglobulins, RheumatoidFactors and Circulating Immune Complexes in their Serum.

The increased B cell compartment in BAFF Tg mice suggested that thelevel of total Ig in the blood of these animals might also be increased.SDS-PAGE, analysis of serum from BAFF Tg mice and control littermatesshowed that the heavy and light chains IgG bands were at least 10 foldmore intense in 3 out of 4 BAFF Tg mice compared to the control sera(FIG. 9A). Likewise, an ELISA determination on the sera from BAFF Tgmice show significantly higher total Ig levels when compared to that ofthe control mice (FIG. 9B).

Despite the high levels seen by SDS-PAGE, the excessively high levels ofIg seen by ELISA determination in some mice, e.g., 697-5, 816-8-3 and823-20, led us to suspect the presence of rheumatoid factors (RF) in thesera, or autoantibodies directed against antigenic determinants on theFc fragment of IgG (Jefferis, 1995). These antibodies could bind to thegoat anti-mouse Ig used to coat the ELISA plates and give erroneouslyhigh values. ELISA plates were coated with normal irrelevant goat Ig andthe binding of BAFF Tg Ig to normal goat Ig was measured. FIG. 9C showsthat sera from most BAFF Tg mice contained Ig reacting with normal goatIg, whereas only two out of 19 control mice exhibited reactivity in thesame assay. These RF were mainly of the IgM, IgA and IgG2a isotypes(data not shown).

Presence of RF can be associated with the presence of high levels ofcirculating immune complexes (CIC) and cryoglobulin in the blood(Jefferis, 1995). To verify whether or not BAFF Tg mice have abnormalserum levels of CIC, a C1q-based binding assay was used to detect CIC inthe 21 BAFF Tg mice analyzed above. Only 5 BAFF Tg showed significantlyhigh levels of CIC when compared to control mice, nonetheless these micecorresponded to the animals having the highest total Ig and rheumatoidfactor levels (FIG. 9D). We also observed precipitate formation whenBAFF Tg mice sera were diluted 1/15 in water but not control seraindicating the presence of cryoglobulin in these mice (data not shown).Thus, in addition to B cell hyperplasia, BAFF Tg mice display severehyperglobulinemia associated with RF and CIC.

Example 5

Some BAFF Tg Mice have High Levels of Anti-Single Stranded (ss) andDouble-Stranded (ds) DNA Autoantibodies.

Initially, we observed kidney abnormalities reminiscent of a lupus-likedisease in two of our founder mice (Table II). The presence of anti-DNAautoantibodies have also been described in SLE patients or the SLE-like(SWR×NZB)F1 (SNF1) mouse (Datta et al., 1987). Anti-ssDNA autoantibodylevels were detected in BAFF Tg mice previously shown to have thehighest level of total serum Ig (FIG. 10A). We analyzed the serum of twoBAFF Tg mice negative for antibodies against ssDNA (697-5 and 816-1-1)and three transgenic mice secreting anti-ssDNA antibodies (820-14,816-8-3 and 820-7) for the presence of anti-dsDNA antibodies in parallelwith five control littermates. BAFF Tg mice also secreted anti-dsDNA,however, the levels of secretion did not always correlate with that ofanti-ssDNA antibodies, as serum from BAFF Tg mouse 697-5 which did notcontain detectable levels of anti-ssDNA antibodies, was clearly positivefor the presence of anti-dsDNA (FIG. 10B). Therefore, BAFF Tg miceshowing the most severe hyperglobulinemia secrete pathological levels ofanti-DNA autoantibodies. Additionally, and also reminiscent of alupus-like problem in these mice we detected immunoglobulin depositionin the kidney of six BAFF Tg mice analyzed (FIG. 10C), three of thesemice did not secrete detectable levels anti-DNA antibodies (data notshown).

Example 6

BAFF Tg Mice have Enlarged B Cell Follicles, Numerous Germinal Centers,Reduced Dendritic Cell Numbers and Increased Plasma Cell Numbers in boththe Spleen and Mesenteric Lymph Nodes (MLN).

BAFF Tg mice had large spleens, MLN (data not shown) and Peyer's patches(FIG. 11). Immunohistochemistry showed the presence of enlarged B cellfollicles and reduced peripheral arteriolar lymphoid sheets (PALS or Tcell area) in BAFF Tg mice (FIG. 12B). Interestingly, few germinalcenters were observed in non-immunized control littermates (and istypical of this colony in general) and those present were small (FIG.12C), whereas BAFF Tg mice possessed numerous germinal centers in theabsence of immunization (FIG. 12D). Staining with anti-CD11c fordendritic cells in the T cell zone and the marginal zone of control mice(FIG. 12E) was considerably reduced in BAFF Tg mice (FIG. 12F).Syndecan-1-positive plasma cells were almost undetectable in the spleenfrom control littermates (FIG. 12G), yet the red pulp of BAFF Tg micewas strongly positive for syndecan-1 (FIG. 12H). Very similarobservations were made for the MLN (FIG. 13). In the MLN of BAFF Tg micethe B cell areas were dramatically expanded (FIG. 13B) in contrast tothe normal node where B cell follicles were easily recognizable at theperiphery of the node under the capsule with a typical paracortical Tcell zone (FIG. 13A). The medulla of MLN from BAFF Tg mice were filledwith syndecan-1 positive cells which presumably are plasma cells (FIG.13H). In conclusion, analysis of secondary lymphoid organs in BAFF Tgmice was consistent with the expanded B cell phenotype showing multiplecellular abnormalities and intense immune activity.

The Following Experimental Procedures were used in Examples 7-13.

Mice and Reagents.

Full length murine BAFF was expressed in transgenic mice using theliver-specific alpha 1 antitrypsin promoter with the Apo E enhancer aspreviously described. (MacKay et al. (1999) J. Exp. Med. 190:1697-1710).C57BL/6 mice were purchased from ARC (Perth, Australia). BAFF transgenicmice are maintained as heterozygotes for the transgene by backcrossingonto C57BL/6 mice. BAFF Tg mice are screened for the presence of thetransgene, both by PCR and southern blot analysis using genomic DNAisolated from 2-3 mm long tail snips. (MacKay et al.). We used animalsfrom two separate lines of BAFF Tg mice issued after 10-12 backcrossingsto C57BL/6. Age-matched negative littermates were used as controls.Animals between 8 and 17 months of age were used. Animals were housedunder conventional barrier protection and handled in accordance with theAnimal Experimentation and Ethic Committee (AEEC), which complies withthe Australian code of practice for the care and use of animals forscientific purposes. Flag-tagged soluble human BAFF (amino acids (aa)83-285) was expressed by E. coli and purified as described previously.(Schneider et al. (1999) J. Exp. Med. 189:1747-1756.) Anti-human BAFFantibodies Buffy-2 (rat IgM), Buffy-5 (Rat IgG1) and A21G3.3 (mouseIgG1, K) were obtained after immunization of rats or mice withrecombinant soluble human BAFF as previously detailed. (Schneider etal.). These antibodies recognize the TNF homology domain of solubleBAFF. The antibody A21G3.3 was purified as follows: 500 ml of mediumfrom hybridoma cultures were diluted 1 to 1 with 0.1 M Sodium Phosphate(pH 7.2) buffer containing 150 mM of NaCl. The diluted media was loadedonto a protein-L column (Clontech, Palo Alto, Calif.) at 1 ml/minute andeluted with 0.1 M Glycine (pH 2.8). The eluted solution was neutralizedwith 1 M Sodium Phosphate buffer (pH 7.2). The peak fractions wereconfirmed by SDS-PAGE. Centricon Plus-20 (Millipore, Bedford, Mass.) wasused to exchange the buffer to PBS and to concentrate the purifiedantibody. The purified A21G3.3 antibody was labelled with 10× mole of EZlink sulfo-NHS-LC Biotin (Pierce, Rockford, Ill.) and incubated at roomtemperature for 30 minutes. The biotinylation reaction was stopped with150 mM glycine. The sample was further purified with a desalting columnto remove the free biotin (Amersham Pharmacia, Uppsala, Sweden). Flowcytometry.

Mice were sacrificed and spleen and submaxillary glands were collected.Spleens and lymph nodes were dissociated by grinding between frostedglass slides (Menzel-Glaser, Braunschweig, Germany). Cells were filteredthrough a 70 μm nylon Cell Strainer (Falcon, Becton Dickinson, FranklinLakes, N.J.) and erythrocytes (only for spleen) removed by osmotic lysiswith red blood cell lysis solution (8.34 mg/ml ammonium chloride, 0.84mg/ml sodium bicarbonate, 1 mM EDTA, pH 8.0). Submaxillary glands werecut into pieces of 2-3 millimeters and incubated in a sterilecollagenase solution (Roche Diagnostics, Mannheim, Germany), 1 mg/ml inPBS (Ca²⁺ and Mg²⁺ free), for 1 h at 37° C., until leukocytes werereleased from the tissue. After digestion, the cell suspension wasfiltered through a 70 μm nylon cell strainer (Falcon) and filtered cellswere washed twice with PBS. Leukocytes obtained from spleens orsubmaxillary glands were resuspended in FACS buffer (1% BSA, 0.05%sodium azide in PBS) at a concentration of 5×10⁶ cells/ml. Surfacestaining was done using various combinations of FITC-, PE-, Cy5- andCychome™-labelled antibodies. Fluorescent-labelled anti-mouse antibodiesanti-CD4 (L3T4), anti-CD8a (Ly-2) anti-CD45R/B220 (RA3-6B2), anti-Ly6-G(GR1), anti-IgD (11-26c.2a), anti-CD11b (Mac1), anti-Ly55 (NK1.1,NKR-P1C), anti-IgM (R6-60.2), anti-CD23 (IgE Fc Receptor, clone B3B4),anti-CD24 (Heat Stable Antigen, 30F1), anti-CD43 (S7), anti-L-selectin(MEL-14), anti-CD1 (1B1) and anti-CD21 (7G6) were supplied by BDPharMingen (San Diego, Calif.). Cy5-conjugated anti-IgM antibody waspurchased from Jackson ImmunoResearch laboratories Inc. (West Grove,Pa.). FITC-labelled antibodies were used diluted 1/100 whereas otherfluorochrome-labelled antibodies were used at 1/200 final dilution. Forflow cytometry we acquired 30,000-100,000 events per sample. Data wascollected on a FACSCalibur flow cytometer and analysed using CELLQues™software (Becton Dickinson).

Immunohistochemistry.

Spleen (removed comma) and submaxillary glands were collected from bothcontrol and BAFF Tg mice. Tissues were either frozen in OCT compound(Tissue-Tek, Sakura Finetek, Torrance, Calif., USA) or fixed in 10%buffered formalin and embedded in paraffin. Biopsies of human parotidglands were processed into paraffin blocks by pathologists at theFlinders Medical center in Adelaide. Paraffin sections, 5 μm thick, werere-hydrated in successive baths of xylene, 100% ethanol and H₂O. Slideswere cooked under pressure in citrate buffer (8.2 mM trisodium citrate,1.7 mM citrate acid, pH 6.0). Slides were either stained withhematoxylin-eosin (H&E) for histologic examination or used forimmunohistochemical staining. Prior to immunohistochemical stainingtissue sections were pre-incubated with human Ig (Sandoz, Basel,Switzerland) 10 μg/ml in TBS-Triton (0.5% Triton) to block non-specificbinding and washed twice with TBS-Triton. Sections were incubated withrat anti-human BAFF (Buffy-2) or an isotype-matched control rat antibody(Jackson ImmunoResearch Laboratories, Inc.), 5 μg/ml for 30 min at roomtemperature, and washed with TBS-Triton. Slides were then incubated withbiotin-labelled rabbit anti-rat Ig (1/100, DAKO (Australia) PTY LTD,Botany, Australia) for 30 min at room temperature, followed byhorseradish peroxidase-labelled (HRP)-Streptavidin (JacksonImmunoResearch Laboratories Inc.) 30 min and visualized using thesubstrate 3,3′ diaminobenzidine (DAB) (Vector laboratories, Inc.,Burlingame, Calif.). Sections were counterstained using hematoxylin(Sigma) and Scott's blueing solution and dehydrated in successive bathsof H₂O, 100% ethanol and xylene. Slides were mounted with cover slipsand Eukitt mounting solution (Calibrated Instruments Inc., Hawthorne,N.Y.). Endogenous peroxidase activity was blocked using 2% hydrogenperoxide in methanol for 20 min before staining with the primaryantibody. Frozen sections of spleen and submaxillary glands weresubjected to immunohistochemical analysis as previously described (32).Biotin-labelled anti-mouse B220 and anti-mouse Syndecan-1 were purchasedfrom BD PharMingen and the staining was detected using HRP-streptavidin(Jackson ImmunoResearch Laboratories Inc.) and visualized using DAB. Allslides were observed under a Leica light microscope and images werecaptured using a Leica DC 200 camera (Leica, Bannockburn, Ill.). Scoringof SS disease activity in mice.

Tissue sections of mouse submaxillary glands stained with H&E wereexamined at 100× under the microscope and scored as previouslydescribed. (White et al. (1974) J. Immunol. 112:178-185). The degree ofinflammatory infiltrates is graded as follows: 1 indicated that 1 to 5foci of mononuclear cells were seen (more than 20 cells per focus), 2indicated that more than 5 foci of mononuclear cells were seen butwithout significant parenchymal destruction, 3 indicated that multipleconfluent foci were seen with moderate degeneration of parenchymaltissue and 4 indicated extensive infiltration of the gland withmononuclear cells and extensive parenchymal destruction.

Measurement of Salivary Flow.

Mice were anaesthetized and injected ip with 50 μg of sterilepilocarpine in PBS (Sigma, St Louis, Mo.) per 100 g body weight. After 4min, saliva was collected for 5 min on a cotton swab. The weight of thecotton swab was measured before and after saliva collection. The amountof saliva collected was normalized to 0 g of saliva per g of bodyweight. Patients with primary SS and sera.

Sera were collected from 41 patients followed between 1995 and 2001 atFlinders Medical Centre who fulfilled at least four of six Europeanconsensus criteria for the diagnosis of primary SS. (Vitali et al.(1993) Arthritis Rheum. 36:340-347. No patient was treated withcorticosteroids or immunosupressive agents. Control sera were collectedfrom 39 healthy donors. Labial salivary gland biopsies with lymphocytefocus scores of >1 per 4 mm² of salivary gland tissue were obtained from4 patients with primary SS (Chisholm, et al. (1968) J. Clin. Pathol.21:656-660), and histologically normal labial salivary gland tissuesobtained from 3 controls.

ELISA Assays for Detection of Human BAFF in Sera from Patients withPrimary SS.

Sera from patients were diluted 1/10 and pre-cleared from human Ig onprotein-A-Sepharose beads (10% beads (pelleted beads) v/v, AmershamPharmacia) overnight at 4° C. ELISA plates (NUNC Nalge International,Rochester, N.Y.) were coated with 2 μg/ml rat anti-human BAFF antibody(Buffy-5), overnight at 4° C. Following blocking, serial dilutions ofthe pre-cleared sera were added, followed by the detection antibody,biotin-conjugated mouse anti-human BAFF (0.5 μg/ml, clone A21G3.3).Alkaline phosphatase (AP)-labelled streptavidin (AP-SA, JacksonImmunoResearch Laboratories Inc.) and the corresponding AP substrateSigma 104 (Sigma) were used for detection. The reaction was stoppedusing 3N NaOH. Plates were read at an OD of 405 nm, and a standard curvewas generated using known quantities of recombinant human BAFF dilutedin human serum and treated as described above for patients' samples.

Statistical Analyses were Done using the StatView Software and ANOVA.

Example 7

BAFF Tg Mice Develop a Sjögren-Like Syndrome with Age.

Following routine dissections of more than 50 BAFF Tg mice and over 20age-matched littermate controls, we observed that many mice over age 13months had enlarged salivary (submaxillary) glands (FIG. 14A).Histological preparations from 22 BAFF Tg mice 12-17 months-old and 7age-matched control mice were examined and scored for disease severity.The scoring system used (White et al.) takes into consideration thenumber of foci of infiltrating leukocytes in the gland and the extent ofdestruction of the parenchymal tissue.

A massive destruction of epithelial duct/acinar cells with smallperiductal foci, as well as large leukocytic infiltrates, was observedin submaxillary glands from BAFF tg animals (FIG. 14B, middle and rightpanel). Although small foci of infiltrating leukocytes were detected inthe submaxillary glands of age-matched control mice, disease gradesremained between 0 and 2, while the majority of BAFF tg mice scored 3 orgreater. Overall, our analysis revealed that 40% of BAFF Tg mice over 12months of age exhibited severe disease (grade 3 or above, FIG. 14C), anincidence which is significantly greater than that observed for controlmice (FIG. 14C). Furthermore, the pathology observed in the BAFF tg micewas independent of the sex of the animals (data not shown). Lacrimalglands were not examined as mice did not develop obvious oculardisorders (keratoconjonctivitis).

Example 8

Presence of Tumor-Like Infiltrates in Salivary Glands of BAFF Tg Mice.

Interestingly. 3 BAFF Tg mice between 13 and 15 months-old, developed alarge submaxillary tumor (over 1 cm of diameter), while no such tumorswere observed in age-matched controls. Histological analysis revealedthat these tumors contained hyperplastic lymphoid tissue composed mainlyof activated B-lymphocytes (FIG. 15D) and numerous germinal centers(FIG. 15B). It is not known whether the tumors arose from infiltrateswithin submaxillary glands or from neighbouring lymph nodes, as noremaining glandular tissue was evident by histology. However, as onlyone submaxillary gland was found in these mice, it is likely that thetumors arose from infiltrates within the gland which ultimatelyoverwhelmed and destroyed all glandular tissue. Unusual aggregates ofcells organised in diffused foci (small lymphocytes in sinusoids) (FIG.15A, B and C) were detected in these tumors (FIG. 15B), as well as inthe large leukocyte infiltrates found in salivary glands of 4independent BAFF Tg mice (two examples are shown in FIGS. 15A and C).Cells in these infiltrates were B-lymphoid cells expressing high levelsof B220 (FIG. 15D), however they were not plasma cells since they werenot positive for the plasma cell marker, syndecan-1 (FIG. 15E). Aconsulting pathologist indicated that because no lympho-epithelialaggregates were seen, the infiltrating B cells could not be definitivelyclassified as malignant. Studies to examine the clonality of theseunusual B cells are in progress to determine whether they are simplytumor-like clusters of lymphoid cells that do not meet criteria formalignancy or cells with real neoplastic potential.

Example 9

Submaxillary Glands from Older BAFF Tg Mice are Infiltrated by a LargeNumber of B-lymphocytes.

Seven BAFF Tg mice that had significantly larger submaxillary glandscompared to age-matched control mice were selected for a detailedassessment of infiltrating cells. One gland from each mouse was digestedwith collagenase, after which mononuclear cells were purified andanalysed by flow cytometry. Absolute counts of various cell types showeda consistent and significant increase in the number of B-lymphocytesinfiltrating submaxillary glands of aging BAFF Tg mice (Table 1).Numbers of T cells, NK cells, macrophages and granulocytes alsoincreased, however a large variation was seen between animals (Table 1).Hematoxylin and eosin staining done on the second gland collected fromeach Tg animal confirmed that the disease score on these tissues was atleast 3 (data not shown). Mice with severe lesions in their submaxillaryglands (disease grade above 3) did not secrete anti-Ro/SSA and/oranti-La/SSB autoantibodies, which are often associated with human SS(data not shown). TABLE 1 Analysis of leukocytes present in salivaryglands of BAFF transgenic mice and control littermates by flowcytometry. Total CD4+ CD8+ Mac-1+ Age Leukocytes B cells T cells T cellsNK cells cells GR1+ cells (months) (×10⁶) (×10⁶) (×10⁶) (×10⁶) (×10⁶)(×10⁶) (×10⁶) Control Littermates Control 1 14 8.4 0.048 — ND 0.04 0.22ND Control 2 14 5.6 0.045 — ND 0.03 0.17 ND Control 3 14 5.9 0.028 — ND0.02 0.23 ND Control 4 15 1.5 0.0014 0.0082 0.0011 0.02 0.57 0.062 Mean± SD 14.25 ± 0.5  5.35 ± 2.8  0.03 ± 0.02 0.027 ± 0.009 0.29 ± 0.18 BAFFTg mice BAFF Tg 1 14 30 1.1 — ND 0.27 0.7 ND BAFF Tg 2 14 31.7 0.14 — ND0.31 1.03 ND BAFF Tg 3 16 42.3 0.6 — ND 0.02 0.23 ND BAFF Tg 4 8 6 0.350.039 0.028 0.13 0.29 0.45 BAFF Tg 5 16 11 4.6 0.084 1.6 0.64 0.27 4.3BAFF Tg 6 17 2.5 0.03 0.027 0.022 0.087 0.17 0.23 BAFF Tg 7 16 22 9.30.34 1.6 1.4 0.5 8.7 Mean ± SD 14.4 ± 3   21 ± 14 2.3 ± 3.5 0.41 ± 0.5 0.46 ± 0.31 p values P < 0.05 P < 0.05Animals were sacrificed and submaxillary glands dissected. Tissues weredigested and leukocytes purified as described in materials and methods.The Total number of leukocytes per gland was counted using anhemocytometer. Cells were stained with anti-B220 (B cells), anti-CD4 andanti-CD8 (T cells), anti-NK1.1 (NK cells), anti-Mac1 (macrophages) andanti-GR1 (granulocytes) fluorochrome-labelled antibodies. Flow cytometryanalysis provided percentage values for each subpopulation# of cells and absolute numbers were calculated relative to the totalnumber of cells collected per gland.ND: not detected,—: not done.Only significant p values are indicated.

Example 10

A Large Proportion of B Cells Infiltrating Submaxillary Glands of BAFFTg Mice have a Marginal Zone (MZ)-Like Phenotype.

A flow cytometric profiling analysis of B cells infiltrating thesubmaxillary glands of BAFF Tg mice revealed two B cell subsets based ontheir level of expression of B220 (FIG. 16A). Analysis of the B-1 B cellpopulation in the B220 low/intermediate (B220^(lo/int)) gate revealed a3-fold increase of B-1b (CD5⁻) B cells and presence of B-1a (CD5⁺) Bcells not detected in control mice (FIG. 16B). In the B220 high(B220^(hi)) gate, cells were further analysed based on their level ofIgM expression. The IgM dull (IgM^(dull)) subset of B cells is presentin both control and BAFF Tg mice, however this population is larger inBAFF Tg mice compared to control animals (FIG. 16B). The IgM^(dull)subset in both control and BAFF Tg mice contains B-2-like cells, whichare CD21^(lo/int), IgD⁺ and CD23⁺ (data not shown). TheB220^(hi)/IgM^(hi) B cell subset is greatly increased in BAFF Tg micecompared to control mice (FIG. 16B). Moreover, the IgM^(hi) B cellsfound in BAFF Tg mice are phenotypically different than IgM^(hi) B cellsfrom control mice. Indeed, the small IgM^(hi) B cell subset detected incontrol mice had characteristics of B-2 B cells (CD21^(lo/int), IgD⁺ andCD23+, add HSA and L-selectin (FIG. 16D, 16E), whereas the largeIgM^(hi) B cell subset of BAFF Tg mice had no counterpart in controlmice and resembled MZ B cells in many respects. These cells wereIgD^(lo), CD21^(hi), CD23^(lol−), CD1^(hi), HSA⁺⁺ (FIG. 16E) andL-selectin^(lo) (FIG. 16D). These cells also express lower levels ofCD43 when compared to B-1 B cells (FIG. 16C). We therefore-refer tothese-cells as M-Z-like-B-cells-because their profile is closer to thatof MZ B cells than to other phenotypically-related B cell subsets suchas B-1 and splenic transitional T1 B cells (FIG. 16F). (Wells et al.(1994) J. Immunol. 153:5503-5515; Amano et al. (1998) J. Immunol.161:1710-1717). However, these cells also share some similarities withB-1b cells, such as low expression of CD43.

Example 11

Older BAFF Tg Mice Exhibit Impaired Saliva Production.

The inflammation seen in the salivary glands of BAFF Tg mice wasreminiscent of the inflammation described for SS patients. Therefore, weaimed to determine if the inflammation in BAFF Tg mice caused animpairment of normal saliva production. To this end, 13 BAFF Tg mice and14 control littermates between 8 and 15.5 months of age receivedpilocarpine, a known stimulant for salivary flow, 4 min prior to thecollection and measurement of saliva. We found that mice between 13 and15 months of age had a significantly reduced production of saliva whencompared to age-matched control animals (FIG. 17A). However, thisdifference was not seen with mice between 8 and 10 months of age (FIG.17B). Interestingly, reduction in saliva flow correlated with thehighest numbers of B-lymphocytes detected in submaxillary glands ofthese Tg animals (data not shown).

Example 12

Elevated Levels of BAFF Detected in the Serum of Patients Suffering fromPrimary Sjögren's Syndrome.

As the Sjögren's-like pathology observed in some BAFF Tg mice showedsimilar features to human SS, we measured BAFF levels in sera ofpatients suffering from this disease. Sera from 41 patients with primarySS and 39 healthy individuals were analysed using a human BAFF-specificELISA assay. This assay showed that at least 15 patients out of 41 (36%)clearly had higher levels of serum BAFF when compared to healthyindividuals (FIG. 18A). Two patients had very high BAFF levels (over 200ng/ml, FIG. 18A). BAFF levels in patients' sera did not correlate withthe levels of total IgG or RF (FIG. 18B and C, respectively). These BAFFlevels also did not correlate with the presence of precipitins such asanti-Ro and/or anti-La (FIG. 18D). We confirmed human BAFF levels inpatients' sera using BAFF-specific immunoprecipitation proceduresfollowed by western blotting techniques (data not shown).

Example 13

Detection of BAFF Expressing Cells in Tissue Sections from Labial Glandsof Patients with SS.

We examined the expression of BAFF at the site of abnormal lymphocyteinfiltrates in biopsies of labial glands from SS patients. A strongBAFF+ signal was detected on leukocytic infiltrates within patienttissues, although clearly not all cells were positive for BAFF withinthe infiltrate (FIG. 18E, right panel). The positive signal detected wasspecific since it could be competed away by the addition of recombinanthuman BAFF (data not shown). The antibody did not stain normal labialsalivary gland tissue (FIG. 18E, bottom left). Preliminary experimentsusing 2-color staining on tissues indicate that infiltratingmacrophages/monocytes are the likely source of BAFF in these inflamedtissues (data not shown).

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1-59. (canceled)
 60. A method of treating a mammal, the methodcomprising administering to a mammal having Sjögren's syndrome acomposition comprising a BAFF blocking agent, thereby reducingimmunoglobulin production or B cell growth in the mammal.
 61. The methodof claim 60, wherein the mammal is a human.
 62. The method of claim 60,wherein the mammal is a mouse.
 63. The method of claim 62, wherein themouse is a BAFF Tg mouse.
 64. The method of claim 60, wherein thesalivary gland of the mammal is infiltrated by MZ-like B cells.
 65. Themethod of claim 60, wherein the BAFF blocking is selected form the groupconsisting of: (a) a soluble BAFF receptor; (b) an antibody against BAFFligand; (c) an antibody against BAFF receptor; and (d) an inoperativeBAFF ligand molecule.
 66. The method of claim 65, wherein soluble BAFFreceptor comprises an immunoglobulin Fc domain.
 67. The method of claim65, wherein the BAFF receptor is BAFF-R.
 68. The method of claim 65,wherein the BAFF receptor is TACI.
 69. The method of claim 65, whereinthe BAFF receptor is BCMA.
 70. The method of claim 67, wherein BAFF-R ishuman.
 71. The method of claim 65, wherein the soluble BAFF receptorcomprises a portion of SEQ ID NO:27 that binds to BAFF.
 72. The methodof claim 71, wherein the soluble BAFF receptor further comprises a humanimmunoglobulin Fc domain.
 73. The method of claim 67, wherein BAFF-R ismurine.
 74. The method of claim 70, wherein the soluble BAFF receptorcomprises a portion of SEQ ID NO:28 that binds to BAFF.
 75. The methodof claim 65, wherein the antibody against BAFF ligand or BAFF receptoris a monoclonal antibody.
 76. The method of claim 75, wherein theantibody is recombinantly produced.
 77. The method of claim 75, whereinthe antibody is a chimeric antibody.
 78. The method of claim 75, whereinthe antibody is a humanized antibody.
 79. The method of claim 75,wherein the antibody comprises human constant domains.
 80. The method ofclaim 75, wherein the antibody is a F(ab′)2 fragment.
 81. The method ofclaim 60, further comprising detecting the level of B cell growth in themammal.
 82. The method of claim 60, further comprising detecting thelevel of immunoglobulin production in the mammal.
 83. The method ofclaim 60, further comprising detecting the levels of B cell growth andimmunoglobulin production in the mammal.
 84. The method of claim 60,further comprising detecting circulating levels of a rheumatoid factorin the mammal.
 85. The method of claim 60, further comprising detectingcirculating levels of anti-DNA autoantibody in the mammal.